Scientific excellence is not an end in itself, but is a crucial tool for efficient translation of new ideas into viable solutions.

Bitter taste receptors (T2Rs) are G protein‑coupled receptors originally detected in the gustatory system. More recently, T2Rs have been shown to be expressed in extra‑oral cells eliciting non‑gustatory functions. Emerging evidence has suggested a potential role for T2R signaling in diverse pathophysiological conditions, including cancer. -The aim of the present study was to evaluate the expression of T2R14 in pancreatic ductal adenocarcinoma (PDAC) and to assess its involvement in the anticancer effects induced by apigenin, a natural ligand of T2R14. For this purpose, T2R14 expression was explored in PDAC tumor tissue and tumor‑derived cell lines. Using the cell lines expressing the highest levels of T2R14, its effects on chemoresponsiveness and migration upon activation with apigenin were investigated in vitro. To the best of our knowledge, the present study was the first to confirm the expression of the T2R family member T2R14 in PDAC. Patients with relatively high levels of T2R14 expression exhibited significantly prolonged overall survival compared with that of patients with low T2R14 expression. Furthermore, novel functions for apigenin were revealed; notably, apigenin was shown to elicit cytotoxic, anti‑migratory and chemosensitizing effects to 5‑fluoruracil (5‑FU) and to 5‑FU, leucovorin, irinotecan and oxaliplatin in pancreatic cancer cells. In conclusion, the present study extended the evidence for the anticancer effects of apigenin and strongly indicated the functional relevance of T2R14 in PDAC, even though their respective underlying pathways appear to be independent of each other.

Several collagen subtypes are involved in pancreatic ductal adenocarcinoma (PDAC) desmoplasia, which constrains therapeutic efficacy. We evaluated collagen type VIII alpha 1 chain (COL8A1), whose function in PDAC is currently unknown. We identified COL8A1 expression in 7 examined PDAC cell lines by microarray analysis, western blotting, and RT‒qPCR. Higher COL8A1 expression occurred in 2 gemcitabine-resistant PDAC cell lines; pancreas tissue (n=15) from LSL-KrasG12D/+; Pdx-1-Cre mice with advanced PDAC predisposition; and PDAC parenchyma and stroma of a patient tissue microarray (n=82). Bioinformatic analysis confirmed higher COL8A1 expression in PDAC patient tissue available from TCGA (n=183), GTEx (n=167), and GEO (n=261) databases. siRNA or lentiviral sh-mediated COL8A1 inhibition in PDAC cells reduced migration, invasion and gemcitabine resistance and resulted in lower cytidine deaminase and thymidine kinase 2 expression and was rescued by COL8A1-secreting cancer-associated fibroblasts (CAFs). The activation of COL8A1 expression involved cJun/AP-1, as demonstrated by CHIP assay and siRNA inhibition. Downstream of COL8A1, activation of ITGB1 and DDR1 receptors and PI3K/AKT and NF-κB signaling occurred, as detected by expression, adhesion and EMSA binding studies. Orthotopic transplantation of PDAC cells with downregulated COL8A1 expression resulted in reduced tumor xenograft growth and lower gemcitabine resistance but was prevented by cotransplantation of COL8A1-secreting CAFs. Most importantly, COL8A1 expression in PDAC patient tissues from our clinic (n=84) correlated with clinicopathological data, and we confirmed these findings by the use of patient data (n=177) from the TCGA database. These findings highlight COL8A1 expression in tumor and stromal cells as a new biomarker for PDAC progression.

Emerging and re-emerging viruses, such as Zaire Ebola virus (EBOV), pose a global threat and require immediate countermeasures, including the rapid development of effective vaccines that are easy to manufacture. Synthetic self-amplifying RNAs (saRNAs) attend to these needs, being safe and strong immune stimulators that can be inexpensively produced in large quantities, using cell-free systems and good manufacturing practice. Here, the first goal was to develop and optimize an anti-EBOV saRNA-based vaccine in terms of its antigen composition and route of administration. Vaccinating mice with saRNAs expressing the EBOV glycoprotein (GP) alone or in combination with the nucleoprotein (NP) elicited antigen-specific immune responses. GP-specific antibodies showed neutralizing activity against EBOV. Strong CD4+ T cell response against NP and GP and CD8+ T cell response against NP were detected by ELISpot assays. Intramuscular vaccination with saRNAs conferred better immune response than intradermal. Finally, mice vaccinated in a prime-boost regimen with saRNAs encoding both GP and NP or with GP alone survived an EBOV infection. In addition, a single dose of GP and NP saRNAs was also protective against fatal EBOV infection. Overall, saRNAs expressing viral antigens represent a promising vaccine platform.

Alphaviruses such as the human pathogenic chikungunya virus (CHIKV) and Ross River virus (RRV) can cause explosive outbreaks raising public health concerns. However, no vaccine or specific antiviral treatment is yet available. We recently established a CHIKV vaccine candidate based on trans-amplifying RNA (taRNA). This novel system consists of a replicase-encoding mRNA and a trans-replicon (TR) RNA encoding the antigen. The TR-RNA is amplified by the replicase in situ. We were interested in determining whether multiple TR-RNAs can be amplified in parallel and if, thus, a multivalent vaccine candidate can be generated. In vitro, we observed an efficient amplification of two TR-RNAs, encoding for the CHIKV and the RRV envelope proteins, by the replicase, which resulted in a high antigen expression. Vaccination of BALB/c mice with the two TR-RNAs induced CHIKV- and RRV-specific humoral and cellular immune responses. However, antibody titers and neutralization capacity were higher after immunization with a single TR-RNA. In contrast, alphavirus-specific T cell responses were equally potent after the bivalent vaccination. These data show the proof-of-principle that the taRNA system can be used to generate multivalent vaccines; however, further optimizations will be needed for clinical application.

Chimeric antigen receptor (CAR) T cells are efficacious in patients with B-cell malignancies, while their activity is limited in patients with solid tumors. We developed a novel heterodimeric TCR-like CAR (TCAR) designed to achieve optimal chain pairing and integration into the T-cell CD3 signaling complex. The TCAR mediated high antigen sensitivity and potent antigen-specific T-cell effector functions in short-term in vitro assays. Both persistence and functionality of TCAR T cells were augmented by provision of costimulatory signals, which improved proliferation in vitro and in vivo. Combination with a nanoparticulate RNA vaccine, developed for in vivo expansion of CAR T cells, promoted tightly controlled expansion, survival, and antitumor efficacy of TCAR T cells in vivo.

Epithelial-to-mesenchymal transition (EMT) renders epithelial cells migratory properties. While epigenetic and splicing changes have been implicated in EMT, the mechanisms governing their crosstalk remain poorly understood. Here we discovered that a C2H2 zinc finger protein, ZNF827, is strongly induced during various contexts of EMT, including in brain development and breast cancer metastasis, and is required for the molecular and phenotypic changes underlying EMT in these processes. Mechanistically, ZNF827 mediated these responses by orchestrating a large-scale remodelling of the splicing landscape by recruiting HDAC1 for epigenetic modulation of distinct genomic loci, thereby slowing RNA polymerase II progression and altering the splicing of genes encoding key EMT regulators in cis. Our findings reveal an unprecedented complexity of crosstalk between epigenetic landscape and splicing programme in governing EMT and identify ZNF827 as a master regulator coupling these processes during EMT in brain development and breast cancer metastasis.

Obesity is a global epidemic and a major predisposing factor for cancer. Increasing evidence shows that obesity-associated stress is a key driver of cancer risk and progression. Previous work has identified the phase-separation organelles, stress granules (SG), as mutant KRAS-dependent mediators of stress adaptation. However, the dependence of tumorigenesis on these organelles is unknown. Here, we establish a causal link between SGs and pancreatic ductal adenocarcinoma (PDAC). Importantly, we uncover that dependence on SGs is drastically heightened in obesity-associated PDAC. Furthermore, we identify a previously unknown regulator and component of SGs, namely, the serine/arginine protein kinase 2 (SRPK2), as a specific determinant of SG formation in obesity-associated PDAC. We show that SRPK2-mediated SG formation in obesity-associated PDAC is driven by hyperactivation of the IGF1/PI3K/mTOR/S6K1 pathway and that S6K1 inhibition selectively attenuates SGs and impairs obesity-associated PDAC development.

(1) Background: A reliable non-invasive distinction between low- and high-risk pancreatic intraductal papillary mucinous neoplasms (IPMN) is needed to effectively detect IPMN with malignant potential. This would improve preventative care and reduce the risk of developing pancreatic cancer and overtreatment. The present study aimed at exploring the presence of autoreactive antibodies in the blood of patients with IPMN of various grades of dysplasia. (2) Methods: A single-center cohort was studied composed of 378 serum samples from patients with low-grade IPMN (n = 91), high-grade IPMN (n = 66), IPMN with associated invasive cancer (n = 30), pancreatic ductal adenocarcinoma (PDAC) stages T1 (n = 24) and T2 (n = 113), and healthy controls (n = 54). A 249 full-length recombinant human protein microarray was used for profiling the serum samples. (3) Results: 14 proteins were identified as potential biomarkers for grade distinction in IPMN, yielding high specificity but mediocre sensitivity. (4) Conclusions: The identified autoantibodies are potential biomarkers that may assist in the detection of malignancy in IPMN patients.

Checkpoint inhibitors (CPI) haverevolutionized the treatment paradigm for advanced solid tumors; however, thereremains an opportunity to improve response rates and outcomes. In preclinicalmodels, 4-1BB costimulation synergizes with CPIs targeting the programmed celldeath protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis byactivating cytotoxic T-cell-mediated antitumor immunity. DuoBody-PD-L1×4-1BB(GEN1046) is an investigational, first-in-class bispecific immunotherapy agentdesigned to act on both pathways by combining simultaneous and complementaryPD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046induced T-cell proliferation, cytokine production, and antigen-specificT-cell-mediated cytotoxicity superior to clinically approved PD-(L)1 antibodiesin human T-cell cultures and exerted potent antitumor activity intransplantable mouse tumor models. In dose escalation of the ongoingfirst-in-human study in heavily pretreated patients with advanced refractorysolid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheralblood consistent with its mechanism of action, manageable safety, and earlyclinical activity [disease control rate: 65.6% (40/61)], including patientsresistant to prior PD-(L)1 immunotherapy.

The arthritogenic alphavirus, chikungunya virus (CHIKV), is now present in almost 100 countries worldwide. Further spread is very likely, which raises public health concerns. CHIKV infections cause fever and arthralgia, which can be debilitating and last for years. Here, we describe a CHIKV vaccine candidate based on trans-amplifying RNA (taRNA). The vaccine candidate consists of two RNAs: a non-replicating mRNA encoding for the CHIKV nonstructural proteins, forming the replicase complex and a trans-replicon (TR) RNA encoding the CHIKV envelope proteins. The TR-RNA can be amplified by the replicase in trans, and small RNA amounts can induce a potent immune response. The TR-RNA was efficiently amplified by the CHIKV replicase in vitro, leading to high protein expression, comparable to that generated by a CHIKV infection. In addition, the taRNA system did not recombine to replication-competent CHIKV. Using a prime-boost schedule, the vaccine candidate induced potent CHIKV-specific humoral and cellular immune responses in vivo in a mouse model. Notably, mice were protected against a high-dose CHIKV challenge infection with two vaccine doses of only 1.5 μg RNA. Therefore, taRNAs are a promising safe and efficient vaccination strategy against CHIKV infections.

Somatic mutations in cancer cells cangenerate tumour-specific neoepitopes, which are recognized by autologous Tcells in the host. As neoepitopes are not subject to central immune toleranceand are not expressed in healthy tissues, they are attractive targets fortherapeutic cancer vaccines. Because the vast majority of cancer mutations areunique to the individual patient, harnessing the full potential of this richsource of targets requires individualized treatment approaches. Manycomputational algorithms and machine-learning tools have been developed toidentify mutations in sequence data, to prioritize those that are more likelyto be recognized by T cells and to design tailored vaccines for every patient.In this Review, we fill the gaps between the understanding of basic mechanismsof T cell recognition of neoantigens and the computational approaches fordiscovery of somatic mutations and neoantigen prediction for cancerimmunotherapy. We present a new classification of neoantigens, distinguishingbetween guarding, restrained and ignored neoantigens, based on how they conferproficient antitumour immunity in a given clinical context. Such context-baseddifferentiation will contribute to a framework that connects neoantigen biologyto the clinical setting and medical peculiarities of cancer, and will enablefuture neoantigen-based therapies to provide greater clinical benefit.

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.

Cancer-associatedgene fusions are a potential source for highly immunogenic neoantigens, but thelack of computational tools for accurate, sensitive identification of personalgene fusions has limited their targeting in personalized cancer immunotherapy. Herewe present EasyFuse, a machine learning computational pipeline for detectingcancer-specific gene fusions in transcriptome data obtained from human cancersamples. EasyFuse predicts personal gene fusions with high precision andsensitivity, outperforming previously described tools. By testingimmunogenicity with autologous blood lymphocytes from patients with cancer, wedetected pre-established CD4+ and CD8+ T cell responses for 10 of 21 (48%) and for 1 of30 (3%) identified gene fusions, respectively. The high frequency of T cellresponses detected in patients with cancer supports the relevance of individualgene fusions as neoantigens that might be targeted in personalizedimmunotherapies, especially for tumors with low mutation burden.

SignificanceFibrosis is a consequence ofmost chronic liver diseases, but currently no approved antifibrotic treatmentis available. M2-type macrophages drive fibrosis progression and preventregression, even when effective causal therapies have been employed. M2-typemacrophages activate a cascade of fibrogenic effector cells and can preventremoval of excess scar tissue. To switch these profibrogenic M2 to fibrolytic(regenerative) macrophages, we developed a pH-degradable, nanogel-baseddelivery system which can be covalently functionalized with themacrophage-repolarizing bisphosphonate alendronate. The nanogels efficientlydeliver the clinically approved drug into hepatic nonparenchymal cells afterintravenous administration. They do not eliminate macrophages but repolarizetheir phenotype and subsequently block fibrosis progression. This approachestablishes a nanotherapeutic delivery platform to treat further M2-typemacrophage-driven diseases, including cancer.

Cancer therapy is an emergentapplication for mRNA therapeutics. While in tumor immunotherapy, mRNA encodingfor tumor-associated antigens is delivered to antigen-presenting cells inspleen and lymph nodes, other therapeutic options benefit from immediate deliveryof mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNAtherapeutics is still a challenge, since, in addition to delivery of the cargoto the tumor, specifics of the targeted cell type as well as its interplay withthe tumor microenvironment are crucial for successful intervention. This studyinvestigated lipoplex nanoparticle-mediated mRNA delivery to spheroid cellculture models of melanoma. Insights into cell-type specific targeting,non-cell-autonomous effects, and penetration capacity in tumor and stroma cellsof the mRNA lipoplex nanoparticles were obtained. It was shown that bothcoculture of different cell types as well as three-dimensional cell growthcharacteristics can modulate distribution and transfection efficiency of mRNAlipoplex formulations. The results demonstrate that three-dimensional coculturespheroids can provide a valuable surplus of information in comparison toadherent cells. Thus, they may represent in vitro models with enhancedpredictivity for the in vivo activity of cancer nanotherapeutics.

Immune checkpoint inhibitors (ICI)targeting the PD-1/PD-L1 axis have changed the treatment paradigm for advancedsolid tumors; however, many patients experience treatment resistance. Inpreclinical models 4-1BB co-stimulation synergizes with ICI by activatingcytotoxic T- and NK-cell-mediated anti-tumor immunity. Here we characterize themechanism of action of a mouse-reactive Fc-inert PD-L1×4-1BB bispecificantibody (mbsAb-PD-L1×4-1BB) and provide proof-of-concept for enhancedanti-tumor activity. In reporter assays mbsAb-PD-L1×4-1BB exhibited conditional4-1BB agonist activity that was dependent on simultaneous binding to PD-L1.mbsAb-PD-L1×4-1BB further blocked the PD-L1/PD-1 interaction independently of4-1BB binding. By combining both mechanisms, mbsAb-PD-L1×4-1BB stronglyenhanced T-cell proliferation, cytokine production and antigen-specificcytotoxicity using primary mouse cells in vitro. Furthermore,mbsAb-PD-L1×4-1BB exhibited potent anti-tumor activity in the CT26 and MC38models in vivo, leading to the rejection of CT26 tumors that wereunresponsive to PD-L1 blockade alone. Anti-tumor activity was associated withincreased tumor-specific CD8+ T cells and reduced regulatory T cells within the tumormicroenvironment and tumor-draining lymph nodes. In immunocompetent tumor-freemice, mbsAb-PD-L1×4-1BB treatment neither induced T-cell infiltration into theliver nor elevated liver enzymes in the blood. Dual targeting of PD-L1 and4-1BB with a bispecific antibody may therefore address key limitations of firstgeneration 4-1BB-agonistic antibodies, and may provide a novel approach toimprove PD-1/PD-L1 checkpoint blockade.

The B.1.1.529 (Omicron) SARS-CoV-2variant has raised global concerns due to its high number of mutations and itsrapid spread. It is of major importance to understand the impact of thisvariant on the acquired and induced immunity. Several preliminary studies havereported the impact of antibody binding and to this date, there are few studieson Omicron’s CD8+ T-cell immune escape.

Gliomas are intrinsic brain tumors witha high degree of constitutive and acquired resistance to standard therapeuticmodalities such as radiotherapy and alkylating chemotherapy. Glioma subtypesare recognized by characteristic mutations. Some of these characteristicmutations have shown to generate immunogenic neoepitopes suitable for targetedimmunotherapy.