Dear Editor, In recent years, treatment with immune checkpoint inhibitors (ICI) has revolutionized cancer therapy. Monoclonal antibodies that block immune checkpoint receptors such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) or its ligand PD-L1 prevent the tumor from suppressing adaptive immune responses.1 Especially PD-L1 is expressed on tumor cells, but also on various healthy cell types.2 When we evaluated a combination therapy of active immunization via dendritic cells (DCs) and ICIs in the context of Merkel cell carcinoma (MCC) in order to improve the efficacy of the treatment, we observed an unexpected inhibitory effect of the anti-PD-L1 ICI Avelumab. In cell culture experiments with the most commonly used type of DCs in clinical trials on therapeutic cancer vaccination,3 the priming capacity of the tumor antigen-loaded human monocyte-derived DCs was reduced in the presence of Avelumab but not Pembrolizumab (anti-PD-1; Figure S1A). Experiments with GFP-expressing DCs showed a disappearance of the DCs in co-cultures with autologous lymphocytes and Avelumab over time (data not shown). However, no direct toxic effect of the antibody against pure DCs was observed (Figure S1B). Moreover, the DCs' ability to stimulate T cell receptor-transfected pure CD8+ T cells was not influenced by the ICI antibodies (Figure S1C). From these unanticipated findings, we concluded that Avelumab, in the presence of autologous lymphocytes, had a detrimental effect on the DCs. In contrast to other approved anti-PD-L1 antibodies like Atezolizumab and Durvalumab, Avelumab contains a constant region (Fc-part) of the IgG1 isotype that is capable of inducing antibody-dependent cellular cytotoxicity (ADCC) against PD-L1 expressing tumor cells, which has been shown to be beneficial in preclinical studies.4 However, healthy cells including DCs also express PD-L1. Therefore, Avelumab could induce an unwanted ADCC reaction against the DCs, as any antibody with a suitable Fc-part that binds efficiently to these cells would do. Flow cytometry experiments showed that monocyte-derived DCs expressed high levels of PD-L1 and that Avelumab, Atezolizumab, and Durvalumab but not Pembrolizumab efficiently bound to the DCs (see Figure 1A). Also, the human primary DC subpopulations cDC1, DC2, and DC3, which are responsible for antigen presentation,5 expressed PD-L1 upon Toll-like receptor 7/8 stimulation with R848 (see Figure 1A). Primarily, two types of cells exert ADCC: macrophages and natural killer (NK) cells. Since the latter are present in substantial numbers in the lymphocyte fractions we used, we examined whether this was the specific cell type that carried out an Avelumab-dependent ADCC against DCs. In a classical chromium-release cytotoxicity assay, we were able to show that in the presence of Avelumab, DCs were efficiently lysed by purified NK cells whereas intermediate lysis was seen when complete peripheral blood mononuclear cells (PBMCs) were used as effector cells (Figure 1B). Consequently, NK-depleted PBMCs showed no lysis. Likewise, there was also no cytotoxicity observed in the control conditions containing no antibody or the anti-PD-1 antibody Pembrolizumab (Figure 1B). The Fc-part-mutated antibodies Durvalumab and Atezolizumab were also not able to induce lysis of the DCs (Figure 1C), although they bound to the DCs to a similar extent (Figure 1A). This clearly shows that Avelumab induced an efficient ADCC reaction by NK cells against autologous DCs. In the experiments described above, monocyte-derived cytokine-matured DCs were used, as these cells are the most commonly used in clinical trials for DC-based therapeutic tumor vaccination. Hence, we examined whether the maturation type and state influenced the susceptibility of DCs to Avelumab-mediated ADCC. We observed that DCs matured with several different stimuli (cytokine cocktail, lipopolysaccharide (LPS), R848, or polyinosinic:polycytidylic acid [poly I:C]) all expressed high levels of PD-L1. Even immature monocyte-derived DCs expressed PD-L1, however to a lower extent. Therefore, the cytotoxicity assay was repeated with purified NK cells and immature DCs, as well as DCs treated with the various maturation stimuli. In the presence of Avelumab all differently matured DCs and even immature DCs were lysed with similarly high efficiency (Figure 1D). This implies that the observed phenomenon may be of general relevance as it seems to apply to all cells expressing a sufficient level of PD-L1. Next to classical ADCC, additional antibody-mediated cytotoxic effects may exist in vivo, like complement-dependent cytotoxicity and antibody-dependent phagocytosis, which may aggravate the effect. In summary, we demonstrated that Avelumab-mediated ADCC via NK-cell activation can lead to the killing of DCs. These findings are of high clinical relevance for combination therapies with Avelumab. Therefore we recommend that patients receiving active immunotherapy like therapeutic vaccination should not simultaneously receive Avelumab treatment because this could result in an inhibitory effect on healthy PD-L1-expressing immune cells—especially when using ex vivo-generated DCs as vaccine. Either a sequential approach with initial vaccination and subsequent Avelumab treatment, or the use of Atezolizumab, Durvalumab, or Pembrolizumab instead of Avelumab is recommended. Avelumab is currently approved for MCC, urothelial carcinoma, and renal cell carcinoma. According to clinicaltrials.gov, Avelumab has been tested in hundreds of clinical trials from phase 1 to phase 3. Most of these combined the antibody with other treatment regimens such as chemotherapy, small molecule inhibitors, other therapeutic antibodies, oncolytic viruses, adoptive cell transfer, and therapeutic vaccination. Two trials used DCs together with Avelumab (NCT03707808 and NCT03152565) and both applied the DCs and the antibody simultaneously. Unfortunately, both did not address the question of whether the antibody had any effect on the DCs. Other trials used other types of vaccines, including peptides, adenoviral vectors, yeast formulations, and again, Avelumab was given at the same time as the vaccine. In a series of discontinued trials (QUILT-3 series) Avelumab was given together with allogenic NK-cells and a recombinant IL-15 superagonist. In one trial with highly progressed MCC patients (NCT03853317), this led to serious adverse events in more than half of the patients, and in another similar trial with pancreatic cancer patients (NCT03136406), one patient reported lymph node pain as an adverse event. In a breast cancer trial (NCT04215146) with paclitaxel and an oncolytic reovirus, the additional application of Avelumab resulted in fewer clinical responses but increased serious adverse events. However, in none of these trials, the effects of the antibody on DCs were examined. Hence, we think that awareness that Avelumab can probably kill important antigen-presenting cells will enable researchers to design more efficient treatment protocols for combination therapies with Avelumab. T.S., F.B., A.C.B., and L.H. performed experiments; T.S., F.B., and J.D. wrote the manuscript; N.C.B. and M.E. provided essential materials; T.S., J.D., N.S., and D.D. supervised experiments; A.C.B., L.H., D.D., N.B., M.E., C.B., and N.S. corrected the manuscript. All authors have read and approved the final manuscript. We appreciate Annett Hamann for helpful technical assistance. We thank the medical staff of the Uniklinikum Erlangen from the Dermatology Department especially Dr. Elias Koch for their support with all blood donations. Furthermore, we would like to thank all voluntary blood donors. We are grateful to Christian Ostalecki for his help in the production of the GFP-labelled Avelumab. We appreciate the working group of Thomas Harrer and especially Katja Schmidt, who supported us with the ELISPOT readouts. The authors declare no conflict of interest. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) via the Research Training Group GRK2504/1 (project number 401821119), research project B2 to Diana Dudziak and B4 to Jan Dörrie. The blood of healthy donors was obtained following informed consent and approval of the institutional review board (Ethics Committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany: Ref. no. 4158). The data generated in this study are available upon request from the corresponding author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Therapies against hematological malignancies using chimeric antigen receptors (CAR)-T cells have shown great potential; however, therapeutic success in solid tumors has been constrained due to limited tumor trafficking and infiltration, as well as the scarcity of cancer-specific solid tumor antigens. Therefore, the enrichment of tumor-antigen specific CAR-T cells in the desired region is critical for improving therapy efficacy and reducing systemic on-target/off-tumor side effects. Here, we functionalized human CAR-T cells with superparamagnetic iron oxide nanoparticles (SPIONs), making them magnetically controllable for site-directed targeting. SPION-loaded CAR-T cells maintained their specific cytolytic capacity against melanoma cells expressing the CAR-specific antigen chondroitin sulfate proteoglycan (CSPG4). Importantly, SPIONs suppressed cytokine release in the loaded CAR-T cells, shifting the cell death phenotype in the tumor cells from pyroptosis to apoptosis. Furthermore, SPION-loaded CAR-T cells could be enriched in a dynamic flow model through an external magnetic field and be detected in MRI. These results demonstrate that lytic cytotoxicity is retained after SPION-functionalization and provides a basis for future site-specific immunotherapies against solid tumors with reduced systemic adverse side effects.

RNA is a substance with various biological functions. It serves as blueprint for proteins and shuttles information from the genes to the protein factories of the cells. However, these factories-the ribosomes-are also composed mainly of RNA, whose purpose is not storing information but enzymatic action. In addition, there is a cornucopia of RNA molecules within our cells that form a complex regulatory network, connected with all aspects of cellular development and maintenance. These non-coding RNAs can be used for diagnostics and therapeutic strategies in cancer. In this chapter we give an overview of recent developments in non-coding RNA-based diagnostics and therapies for cutaneous melanoma. It is not meant to be comprehensive; however, it describes examples based on some of the most recent publications in this field.

The RNA world is wide, and besides mRNA, there is a variety of other RNA types, such as non-coding (nc)RNAs, which harbor various intracellular regulatory functions. This review focuses on small interfering (si)RNA and micro (mi)RNA, which form a complex network regulating mRNA translation and, consequently, gene expression. In fact, these RNAs are critically involved in the function and phenotype of all cells in the human body, including malignant cells. In cancer, the two main targets for therapy are dysregulated cancer cells and dysfunctional immune cells. To exploit the potential of mi- or siRNA therapeutics in cancer therapy, a profound understanding of the regulatory mechanisms of RNAs and following targeted intervention is needed to re-program cancer cells and immune cell functions in vivo. The first part focuses on the function of less well-known RNAs, including siRNA and miRNA, and presents RNA-based technologies. In the second part, the therapeutic potential of these technologies in treating cancer is discussed, with particular attention on manipulating tumor-associated immune cells, especially tumor-associated myeloid cells.

Background The composition of the tumor microenvironment in solid tumors is of crucial importance for the prognosis and clinical outcome of patients with solid cancers (1). Infiltration of CD8+ T cells into the tumor can improve the prognosis and treatment options of patients. Adoptive T cell therapy is intended to increase the number of CD8+ T cells in the tumor. However, only a fraction of cancer patients benefit from this option, partially because the T cells do not effectively reach the tumor (2). We developed citrate-coated superparamagnetic iron oxide nanoparticles (SPIONs) for the loading of T cells to make them magnetically controllable (3,4). After intra-arterial application and magnetic enrichment in the tumor region, SPION-loaded T cells must pass through the vessel wall to reach the tumor and they must retain antigen-specific effector functions to fight the tumor. This study investigated the effects of SPION loading on primary human T cells, particularly on antigen-specific effector functions and their cellular migration capacity (5). Materials and Methods T cells were freshly isolated from human whole blood and subsequently loaded with SPIONs for 4 h. Unloaded T cells served as controls. Using a Boyden-Chamber-based assay, we acquired information about the ability of T cell to migrate towards a CXCL12-gradient. Furthermore, the tethering and attachment of T cells on an endothelial cell monolayer was investigated by fluorescence microscopy. The deformability upon SPION-loading was investigated using Real-Time Deformability Cytometry (RT-DC). Antigen-specific effector functions were examined after stimulation via an introduced exogenous T cell receptor (TCR) specific for the melanoma antigen MelanA or the endogenous TCR specific for the cytomegalovirus antigen pp65. Results SPION-loading had no effect on the attachment of T cells to an endothelial monolayer, however, the chemotactic migration was reduced by SPIONs, which was cancelled out by magnetic attraction. RT-DC ruled out stiffening of the cells due to nanoparticle loading, which is important for squeezing through the vessel walls during transmigration. Lastly, we observed no alterations in antigen-specific effector functions regarding proliferation, expression of activation markers, cytokine secretion, or tumor cell killing after antigen-specific activation mediated by endo- or exogenous TCRs. Conclusions In sum, we showed that SPION loading did not impair cellular mechanics or antigen-specific effector functions. With regard to cell transmigration, possible negative effects of SPION-loading on the T cells were compensated by magnetic attraction. These results underline the potential of SPIONs for the enrichment of T cells in the tissue of solid tumors through magnetic attraction. References Giraldo NA, et al. Br J Cancer 2019. Morotti, M, et al. Br J Cancer 2021. Boosz P, et al. Cancers 2021. Mühlberger M, et al. Int J Nanomedicine 2019. Pfister F, et al. Front Immunol 2023. L.R. Carnell: None. S. Knorr: None. F. Pfister: None. J. Dörrie: None. N. Schaft: None. C. Alexiou: None. C. Janko: None.

The large T antigen (LT) of the Merkel cell polyomavirus (MCPyV) is crucial for Merkel cell carcinoma (MCC), a rare but very aggressive form of neuroendocrine skin cancer. The clonal integration of MCPyV DNA into the host genome is a signature event of this malignancy. The resulting expression of oncogenes, including the small T (sT) antigen and a truncated form of the LT (truncLT), directly contribute to carcinogenesis. The truncation of the C-terminus of LT prevents the virus from replicating due to the loss of the origin binding domain (OBD) and the helicase domain. This precludes cytopathic effects that would lead to DNA damage and ultimately cell death. At the same time, the LxCxE motif in the N-terminus is retained, allowing truncLT to bind the retinoblastoma protein (pRb), a cellular tumor suppressor. The continuously inactivated pRb promotes cell proliferation and tumor development. truncLT exerts several classical functions of an oncogene: altering the host cell cycle, suppressing innate immune responses to viral DNA, causing immune escape, and shifting metabolism in favor of cancer cells. Given its central role in MCC, the LT is a major target for therapeutic interventions with novel approaches, such as immune checkpoint inhibition, T cell-based immunotherapy, and cancer vaccines.

BackgroundTumor-associated antigens and their derived peptides constitute an opportunity to design off-the-shelf mainline or adjuvant anti-cancer immunotherapies for a broad array of patients. A performant and rational antigen selection pipeline would lay the foundation for immunotherapy trials with the potential to enhance treatment, tremendously benefiting patients suffering from rare, understudied cancers.MethodsWe present an experimentally validated, data-driven computational pipeline that selects and ranks antigens in a multipronged approach. In addition to minimizing the risk of immune-related adverse events by selecting antigens based on their expression profile in tumor biopsies and healthy tissues, we incorporated a network analysis-derived antigen indispensability index based on computational modeling results, and candidate immunogenicity predictions from a machine learning ensemble model relying on peptide physicochemical characteristics.ResultsIn a model study of uveal melanoma, Human Leukocyte Antigen (HLA) docking simulations and experimental quantification of the peptide–major histocompatibility complex binding affinities confirmed that our approach discriminates between high-binding and low-binding affinity peptides with a performance similar to that of established methodologies. Blinded validation experiments with autologous T-cells yielded peptide stimulation-induced interferon-γ secretion and cytotoxic activity despite high interdonor variability. Dissecting the score contribution of the tested antigens revealed that peptides with the potential to induce cytotoxicity but unsuitable due to potential tissue damage or instability of expression were properly discarded by the computational pipeline.ConclusionsIn this study, we demonstrate the feasibility of the de novo computational selection of antigens with the capacity to induce an anti-tumor immune response and a predicted low risk of tissue damage. On translation to the clinic, our pipeline supports fast turn-around validation, for example, for adoptive T-cell transfer preparations, in both generalized and personalized antigen-directed immunotherapy settings.

With the advent of immunotherapeutics, a new era in the combat against cancer has begun. Particularly promising are neo-epitope-targeted therapies as the expression of neo-antigens is tumor-specific. In turn, this allows the selective targeting and killing of cancer cells whilst healthy cells remain largely unaffected. So far, many advances have been made in the development of treatment options which are tailored to the individual neo-epitope repertoire. The next big step is the achievement of efficacious “off-the-shelf” immunotherapies. For this, shared neo-epitopes propose an optimal target. Given the tremendous potential, a thorough understanding of the underlying mechanisms which lead to the formation of neo-antigens is of fundamental importance. Here, we review the various processes which result in the formation of neo-epitopes. Broadly, the origin of neo-epitopes can be categorized into three groups: canonical, noncanonical, and viral neo-epitopes. For the canonical neo-antigens that arise in direct consequence of somatic mutations, we summarize past and recent findings. Beyond that, our main focus is put on the discussion of noncanonical and viral neo-epitopes as we believe that targeting those provides an encouraging perspective to shape the future of cancer immunotherapeutics.

Background Different hematological cancer types have shown promising responses to autologous chimeric antigen receptor (CAR)-T cell therapy. However, the efficacy of this treatment in solid tumors is hindered by challenges such as poor tumor infiltration, long-term retention of CAR-T cells, and systemic side effects. To address these limitations, we developed citrate-coated superparamagnetic iron oxide nanoparticles (SPIONs), which have the ability to adhere to and be taken up by T cells, thereby enabling the control of CAR-T cells through an external magnetic field (1-3). In future, magnetic guidability should help to enrich CAR-T cells in the tumor microenvironment, leading to site-specific anti-tumor responses. This study aims to investigate the influence of SPION-loading of CAR-T cells on their efficacy in anti-tumor cell responses in vitro. Materials and Methods T cells were isolated from peripheral blood of healthy donors and received mRNA encoding a chondroitin sulfate proteoglycan 4 (CSGP4)-specific CAR via electroporation (4, 5). The cells were then incubated with SPIONs for 4h to magnetically functionalize them. Subsequently, T cells were co-incubated with melanoma tumor cells expressing CSGP4 on their surface. Afterwards, the cells were analyzed for their antigen-specific anti-tumor responses and compared to non-loaded CAR-T cells or CSGP4-negative tumor cells by flow cytometry. Additionally, tumor cell lysis was investigated via impedance-based monitoring of cell viability and microscopic analysis of the dissolution of three-dimensional tumor spheroids. Results We observed that SPION-loading did not affect the expression of activation markers, differentiation, or proliferation of CAR-T cells. Furthermore, SPION-loaded CAR-T cells retained their capability for antigen-specific tumor cell lysis over multiple days. Additionally, these CAR-T cells demonstrated the ability to be controlled by an external magnetic field, as well as infiltrating and dissolving tumor spheroids. Conclusions In summary, we demonstrated that SPION-loading did not compromise the functionality of CAR-T cells, as they were still able to perform the investigated effector functions with similar efficacy as the non-loaded control CAR-T cells. These findings underscore the potential of SPIONs in enhancing site-specific anti-tumor responses of CAR-T cells in the therapy of solid cancers in the future. References Mühlberger et al. J. Magn. 2019. Boosz et al. Cancers. 2021. Pfister et al. Front Immunol. 2023. Krug et al. Cancer Immunol Immunother. 2015. Harrer al. Int J Mol Sci. 2019. F. Pfister: None. L. Löffler: None. L.R. Carnell: None. P. Boosz: None. J. Dörrie: None. N. Schaft: None. C. Alexiou: None. C. Janko: None.

This report details a case of pancreatic cancer with liver metastasis that exhibited a positive immune response to personalized immunization therapy. Our study involved the identification of neoantigens and their corresponding immunogenic peptides using an in-house bioinformatic pipeline. This process included the identification of somatic mutations through DNA/RNA sequencing of solid tumor tissue and blood liquid biopsy. Computational prediction techniques were then employed to identify novel epitopes, followed by the design and manufacture of patient-specific immunization peptides. In combination with standard-of-care chemotherapy, the patient received a sequence of 5 biweekly prime injections followed by 2 boost injections 2 and 5 months later. The peptides were emulsified in Montanide and the injection-site was conditioned with nivolumab and imiquimod. The combined regimen of peptide immunization and chemotherapy resulted in a notable decline in CA19-9 tumor marker levels following both prime and boost applications. Subsequent MRI assessments revealed a reduction in the size of liver metastases several months post-immunization initiation. Importantly, the patient showed and improved overall survival and reported an improved quality of life without experiencing significant treatment-related adverse effects. This case underscores the potential benefits of personalized peptide-based immunization as an adjunctive therapy in the treatment of advanced pancreatic cancer, showcasing promising outcomes in tumor marker reduction, tumor shrinkage, and enhanced patient well-being.

The development of chimeric antigen receptor T cells (CAR-T cells) has marked a new era in cancer immunotherapy. Based on a multitude of durable complete remissions in patients with hematological malignancies, FDA and EMA approval was issued to several CAR products targeting lymphoid leukemias and lymphomas. Nevertheless, about 50% of patients treated with these approved CAR products experience relapse or refractory disease necessitating salvage strategies. Moreover, in the vast majority of patients suffering from solid tumors, CAR-T-cell infusions could not induce durable complete remissions so far. Crucial obstacles to CAR-T-cell therapy resulting in a priori CAR-T-cell refractory disease or relapse after initially successful CAR-T-cell therapy encompass antigen shutdown and CAR-T-cell dysfunctionality. Antigen shutdown predominately rationalizes disease relapse in hematological malignancies, and CAR-T-cell dysfunctionality is characterized by insufficient CAR-T-cell proliferation and cytotoxicity frequently observed in patients with solid tumors. Thus, strategies to surmount those obstacles are being developed with high urgency. In this review, we want to highlight different approaches to combine CAR-T cells with drugs, such as small molecules and antibodies, to pharmacologically boost CAR-T-cell therapy. In particular, we discuss how certain drugs may help to counteract antigen shutdown and CAR-T-cell dysfunctionality in both hematological malignancies and solid tumors.

Contains further information on raw data, its sources and processing (NGS pipeline, tissue lists and selection). We also provide supporting information on the selected genes and their expression patterns in the relevant tissues.

Dendritic cells (DCs) are major regulators of innate and adaptive immune responses. DCs can be classified into plasmacytoid DCs and conventional DCs (cDCs) type 1 and 2. Murine and human cDC1 share the mRNA expression of XCR1. Murine studies indicated a specific role of the XCR1-XCL1 axis in the induction of immune responses. Here, we describe that human cDC1 can be distinguished into XCR1(-) and XCR1(+) cDC1 in lymphoid as well as nonlymphoid tissues. Steady-state XCR1(+) cDC1 display a pre-activated phenotype compared to XCR1(-) cDC1. Upon stimulation, XCR1(+) cDC1, but not XCR1(-) cDC1, secreted high levels of inflammatory cytokines as well as chemokines. This was associated with enhanced activation of NK cells mediated by XCR1(+) cDC1. Moreover, XCR1(+) cDC1 excelled in inhibiting replication of Influenza A virus. Further, under DC differentiation conditions, XCR1(-) cDC1 developed into XCR1(+) cDC1. After acquisition of XCR1 expression, XCR1(-) cDC1 secreted comparable level of inflammatory cytokines. Thus, XCR1 is a marker of terminally differentiated cDC1 that licenses the antiviral effector functions of human cDC1, while XCR1(-) cDC1 seem to represent a late immediate precursor of cDC1.

Background:Immunotherapy of cancer is an emerging field with the potential to improve long-term survival. Thus far, adoptive transfer of tumor-specific T cells represents an effective treatment option for tumors of the hematological system such as lymphoma, leukemia or myeloma. However, in solid tumors, treatment efficacy is low owing to the immunosuppressive microenvironment, on-target/off-tumor toxicity, limited extravasation out of the blood vessel, or ineffective trafficking of T cells into the tumor region. Superparamagnetic iron oxide nanoparticles (SPIONs) can make cells magnetically controllable for the site-specific enrichment.Methods:In this study, we investigated the influence of SPION-loading on primary human T cells for the magnetically targeted adoptive T cell therapy. For this, we analyzed cellular mechanics and the T cell response after stimulation via an exogenous T cell receptor (TCR) specific for the melanoma antigen MelanA or the endogenous TCR specific for the cytomegalovirus antigen pp65 and compared them to T cells that had not received SPIONs.Results:SPION-loading of human T cells showed no influence on cellular mechanics, therefore retaining their ability to deform to external pressure. Additionally, SPION-loading did not impair the T cell proliferation, expression of activation markers, cytokine secretion, and tumor cell killing after antigen-specific activation mediated by the TCR.Conclusion:In summary, we demonstrated that SPION-loading of T cells did not affect cellular mechanics or the functionality of the endogenous or an exogenous TCR, which allows future approaches using SPIONs for the magnetically enrichment of T cells in solid tumors.

The treatment of cancer was revolutionized within the last two decades by utilizing the mechanism of the immune system against malignant tissue in so-called cancer immunotherapy. Two main developments boosted cancer immunotherapy: 1) the use of checkpoint inhibitors, which are characterized by a relatively high response rate mainly in solid tumors; however, at the cost of serious side effects, and 2) the use of chimeric antigen receptor (CAR)-T cells, which were shown to be very efficient in the treatment of hematologic malignancies, but failed to show high clinical effectiveness in solid tumors until now. In addition, active immunization against individual tumors is emerging, and the first products have reached clinical approval. These new treatment options are very cost-intensive and are not financially compensated by health insurance in many countries. Hence, strategies must be developed to make cancer immunotherapy affordable and to improve the cost-benefit ratio. In this review, we discuss the following strategies: 1) to leverage the antigenicity of “cold tumors” with affordable reagents, 2) to use microbiome-based products as markers or therapeutics, 3) to apply measures that make adoptive cell therapy (ACT) cheaper, e.g., the use of off-the-shelf products, 4) to use immunotherapies that offer cheaper platforms, such as RNA- or peptide-based vaccines and vaccines that use shared or common antigens instead of highly personal antigens, 5) to use a small set of predictive biomarkers instead of the “sequence everything” approach, and 6) to explore affordable immunohistochemistry markers that may direct individual therapies.

Dendritic cells (DCs) can be used for therapeutic vaccination against cancer. The success of this therapy depends on efficient tumor-antigen presentation to cytotoxic T lymphocytes (CTLs) and the induction of durable CTL responses by the DCs. Therefore, simulation of such a biological system by computational modeling is appealing because it can improve our understanding of the molecular mechanisms underlying CTL induction by DCs and help identify new strategies to improve therapeutic DC vaccination for cancer. Here, we developed a multi-level model accounting for the life cycle of DCs during anti-cancer immunotherapy. Specifically, the model is composed of three parts representing different stages of DC immunotherapy – the spreading and bio-distribution of intravenously injected DCs in human organs, the biochemical reactions regulating the DCs’ maturation and activation, and DC-mediated activation of CTLs. We calibrated the model using quantitative experimental data that account for the activation of key molecular circuits within DCs, the bio-distribution of DCs in the body, and the interaction between DCs and T cells. We showed how such a data-driven model can be exploited in combination with sensitivity analysis and model simulations to identify targets for enhancing anti-cancer DC vaccination. Since other previous works show how modeling improves therapy schedules and DC dosage, we here focused on the molecular optimization of the therapy. In line with this, we simulated the effect in DC vaccination of the concerted modulation of combined intracellular regulatory processes and proposed several possibilities that can enhance DC-mediated immunogenicity. Taken together, we present a comprehensive time-resolved multi-level model for studying DC vaccination in melanoma. Although the model is not intended for personalized patient therapy, it could be used as a tool for identifying molecular targets for optimizing DC-based therapy for cancer, which ultimately should be tested in in vitro and in vivo experiments.

Due to their ability to trigger strong immune responses, adenoviruses (HAdVs) in general and the serotype5 (HAdV-5) in particular are amongst the most popular viral vectors in research and clinical application. However, efficient transduction using HAdV-5 is predominantly achieved in coxsackie and adenovirus receptor (CAR)-positive cells. In the present study, we used the transduction enhancer LentiBOOST® comprising the polycationic Polybrene to overcome these limitations. Using LentiBOOST®/Polybrene, we yielded transduction rates higher than 50% in murine bone marrow-derived dendritic cells (BMDCs), while maintaining their cytokine expression profile and their capability to induce T-cell proliferation. In human dendritic cells (DCs), we increased the transduction rate from 22% in immature (i)DCs or 43% in mature (m)DCs to more than 80%, without inducing cytotoxicity. While expression of specific maturation markers was slightly upregulated using LentiBOOST®/Polybrene on iDCs, no effect on mDC phenotype or function was observed. Moreover, we achieved efficient HAdV5 transduction also in human monocytes and were able to subsequently differentiate them into proper iDCs and functional mDCs. In summary, we introduce LentiBOOST® comprising Polybrene as a highly potent adenoviral transduction agent for new in-vitro applications in a set of different immune cells in both mice and humans.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Uveal melanoma (UM) is an orphan disease with a mortality of 80% within one year upon the development of metastatic disease. UM does hardly respond to chemotherapy and kinase inhibitors and is largely resistant to checkpoint inhibition. Hence, further therapy approaches are urgently needed. To improve clinical outcome, we designed a trial employing the 3rd generation personalized IKKβ-matured RNA-transfected dendritic cell (DC) vaccine which primes T cells and in addition activates NK cells. This ongoing phase I trial [NCT04335890 (www.clinicaltrials.gov), Eudract: 2018-004390-28 (www.clinicaltrialsregister.eu)] investigates patients with treatment-naive metastatic UM. Monocytes are isolated by leukapheresis, differentiated to immature DCs, matured with a cytokine cocktail, and activated via the NF-κB pathway by electroporation with RNA encoding a constitutively active mutant of IKKβ. Three types of antigen-RNA are co-electroporated: i) amplified mRNA of the tumor representing the whole transcriptome, ii) RNA encoding driver mutations identified by exome sequencing, and iii) overexpressed non-mutated tumor antigens detected by transcriptome sequencing. This highly personalized DC vaccine is applied by 9 intravenous infusions in a staggered schedule over one year. Parallel to the vaccination, standard therapy, usually an immune checkpoint blockade (ICB) as mono (anti-PD-1) or combined (anti-CTLA4 and anti-PD-1) regimen is initiated. The coordinated vaccine-induced immune response encompassing tumor-specific T cells and innate NK cells should synergize with ICB, perhaps resulting in measurable clinical responses in this resistant tumor entity. Primary outcome measures of this trial are safety, tolerability and toxicity; secondary outcome measures comprise overall survival and induction of antigen-specific T cells.

The addition of CAR-T cells to the armamentarium of immunotherapy revigorated the field of oncology by inducing long-lasting remissions in patients with relapsing/refractory hematological malignancies. Nevertheless, in the lion's share of patients diagnosed with solid tumors, CAR-T-cell therapy so far failed to demonstrate satisfactory anti-tumor activity. A crucial cause of resistance against the antigen-specific attack of CAR-T cells is predicated on the primary or secondary absence of suitable target antigens. Thus, the necessity to create a broad repertoire of different target antigens is vital. We aimed to evaluate the potential of the well-established melanoma antigen chondroitin sulfate proteoglycan 4 (CSPG4) as an inducible antigen in ovarian cancer cells, using CSPG4-negative SKOV-3 ovarian cancer cells as a model. Based on the hypomethylating activity of the FDA-approved drug decitabine, we refined a protocol to upregulate CSPG4 in the majority of decitabine-treated SKOV-3 cells. CSPG4-specific CAR-T cells generated by mRNA-electroporation showed CSPG4-directed cytokine secretion and cytotoxicity towards decitabine-treated SKOV-3. Another ovarian cancer cell line (Caov-3) and the neoplastic cell line 293T behaved similar. In aggregate, we generated proof-of-concept data paving the way for the further exploration of CSPG4 as an inducible antigen for CAR-T cells in ovarian cancer.