Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand ...for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay’s ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay’s combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.
Treating patients who have cancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with ...treatment-refractory tumours
. We are testing melanoma FixVac (BNT111)-an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma-in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4
and CD8
T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination.
T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome ...vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.
Triple-negative breast cancer (TNBC) is a high medical need disease with limited treatment options. CD8+ T cell-mediated immunotherapy may represent an attractive approach to address TNBC. The ...objectives of this study were to assess the expression of CXorf61 in TNBCs and healthy tissues and to evaluate its capability to induce T cell responses. We show by transcriptional profiling of a broad comprehensive set of normal human tissue that CXorf61 expression is strictly restricted to testis. 53% of TNBC patients express this antigen in at least 30% of their tumor cells. In CXorf61-negative breast cancer cell lines CXorf61 expression is activated by treatment with the hypomethylating agent 5-aza-2'-deoxycytidine. By vaccination of HLA-A*02-transgenic mice with CXorf61 encoding RNA we obtained high frequencies of CXorf61-specific T cells. Cloning and characterization of T cell receptors (TCRs) from responding T cells resulted in the identification of the two HLA-A*0201-restricted T cell epitopes CXorf6166-74 and CXorf6179-87. Furthermore, by in vitro priming of human CD8+ T cells derived from a healthy donor recognizing CXorf6166-74 we were able to induce a strong antigen-specific immune response and clone a human TCR recognizing this epitope. In summary, our data confirms this antigen as promising target for T cell based therapies.
Highlights • TPTE autoantibodies can be found in the sera of 13.4% of lung cancer patients by CrELISA. • Sensitivity and Specificy for diagnosing lung cancer are modest. • TPTE autoantibodies are ...associated with prolonged survival.
The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and ...infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-specific CD8(+) or CD4(+) T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8(+) and CD4(+) T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes.
Drug delivery to the brain is limited for most pharmaceuticals by the blood-brain barrier (BBB) where claudin-5 dominates the paraendothelial tightening. For circumventing the BBB, we identified the ...compound M01 as a claudin-5 interaction inhibitor. M01 causes transient permeabilisation of the BBB depending on the concentration of small molecules in different cell culture models within 3 to 48 h. In mice, brain uptake of fluorescein peaked within the first 3 h after M01 injection and normalised within 48 h. Compared to the cytostatic paclitaxel alone, M01 improved delivery of paclitaxel to mouse brain and reduced orthotopic glioblastoma growth. Results on interactions of M01 with claudin-5 were incorporated into a binding model which suggests association of its aromatic parts with highly conserved residues of the extracellular domain of claudin-5 and adjacent transmembrane segments. Our results indicate the following mode of action: M01 preferentially binds to the extracellular claudin-5 domain, which weakens trans-interactions between adhering cells. Further decrease in membranous claudin-5 levels due to internalization and transcriptional downregulation enables the paracellular passage of small molecules. In summary, the first small molecule is introduced here as a drug enhancer, which specifically permeabilises the BBB for a sufficient interval for allowing neuropharmaceuticals to enter the brain.
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Abstract
In der Grundlagenforschung wird die Testung heterogener Katalysatoren normalerweise mit sehr kleinen Probenmengen durchgeführt. Eine Übertragung der ermittelten Ergebnisse hinsichtlich ...Produktivität und Standzeit auf den technischen Maßstab ist nur sehr eingeschränkt möglich. Es wurde ein neues praxisnahes Testsystem für heterogene Katalysatoren entwickelt. Es kann sowohl in Festbett‐Fahrweise als auch in Slurry‐Fahrweise in einem weiten Temperatur‐ und Druckbereich betrieben werden. Das System wurde für die Synthese höherer Alkohole und die einstufige Dimethylether‐Synthese erfolgreich eingesetzt.
Abstract
In fundamental heterogeneous catalyst development, testing is usually performed with rather small amounts of catalyst. Transferring the results in terms of performance and catalyst lifetime to industrial relevant pilot‐scale is hardly feasible. Thus, a new modular test system has been developed which allows large‐scale testing of heterogeneous catalysts in fixed‐bed or slurry mode, enabling a wide range of test conditions with regard to particle size, gas composition, pressure, and reaction temperature. The test system has been used for higher alcohol synthesis and one‐step dimethyl ether synthesis.
In der Grundlagenforschung wird die Testung heterogener Katalysatoren normalerweise mit sehr kleinen Probenmengen durchgeführt. Eine Übertragung der ermittelten Ergebnisse hinsichtlich Produktivität ...und Standzeit auf den technischen Maßstab ist nur sehr eingeschränkt möglich. Es wurde ein neues praxisnahes Testsystem für heterogene Katalysatoren entwickelt. Es kann sowohl in Festbett‐Fahrweise als auch in Slurry‐Fahrweise in einem weiten Temperatur‐ und Druckbereich betrieben werden. Das System wurde für die Synthese höherer Alkohole und die einstufige Dimethylether‐Synthese erfolgreich eingesetzt.
In fundamental heterogeneous catalyst development, testing is usually performed with rather small amounts of catalyst. Transferring the results in terms of performance and catalyst lifetime to industrial relevant pilot‐scale is hardly feasible. Thus, a new modular test system has been developed which allows large‐scale testing of heterogeneous catalysts in fixed‐bed or slurry mode, enabling a wide range of test conditions with regard to particle size, gas composition, pressure, and reaction temperature. The test system has been used for higher alcohol synthesis and one‐step dimethyl ether synthesis.
Vorgestellt wird ein neues System zur praxisnahen Testung von heterogenen Katalysatoren. Das System soll die Lücke zwischen der Katalysatortestung im Labormaßstab und im technischen Maßstab schließen. Im Festbett‐ und im Slurry‐Reaktor wurden Katalysatoren für die Synthesegaskonversion zu Alkoholen und Dimethylether getestet.
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