25 results on '"Prommersberger S"'
Search Results
2. Volumetric quantification of epicardial fat using semiautomated atrial CT segmentation in patients with atrial fibrillation: the Prospective AFAT study
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Erhard, N, primary, Prommersberger, S, additional, Neuner, B, additional, Bahlke, F, additional, Englert, F, additional, Popa, M A, additional, Krafft, H, additional, Abdiu, E, additional, Reents, T, additional, Lennerz, C, additional, Telishevska, M, additional, Lengauer, S, additional, Hessling, G, additional, Deisenhofer, I, additional, and Bourier, F, additional
- Published
- 2024
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3. A new method to monitor antigen-specific CD8+ T cells, avoiding additional target cells and the restriction to human leukocyte antigen haplotype
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Prommersberger, S, Höfflin, S, Schuler-Thurner, B, Schuler, G, Schaft, N, and Dörrie, J
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- 2015
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4. High antileukemic efficiency of CD19-CAR NK cells engineered with Sleeping Beauty transposon vectors
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Bexte, T, additional, Botezatu, L, additional, Miskey, C, additional, Reindl, LM, additional, Campe, J, additional, Mertlitz, S, additional, Gebel, V, additional, Schubert, R, additional, Cremer, A, additional, Rettinger, E, additional, Prommersberger, S, additional, Penack, O, additional, Wels, WS, additional, Hudecek, M, additional, Ivics, Z, additional, and Ullrich, E, additional
- Published
- 2022
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5. Mutated tumor antigens as targets for cancer immunotherapy: P157
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Prommersberger, S., Höfflin, S., Gross, S., Schuler, G., Dörrie, J., and Schaft, N.
- Published
- 2012
6. Genomic Analyses of SLAMF7 CAR-T Cells Manufactured by Sleeping Beauty Transposon Gene Transfer for Immunotherapy of Multiple Myeloma
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M Machwirth, Prommersberger S, Halvard Bonig, Reiser M, Michael Hudecek, Beckmann J, Maximilian Amberger, Zoltán Ivics, Csaba Miskey, and Hermann Einsele
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Transposable element ,0303 health sciences ,T cell ,Context (language use) ,Computational biology ,Biology ,Sleeping Beauty transposon system ,Chimeric antigen receptor ,3. Good health ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Antigen ,030220 oncology & carcinogenesis ,medicine ,media_common.cataloged_instance ,European union ,Transposase ,030304 developmental biology ,media_common - Abstract
Widespread treatment of human diseases with gene therapies necessitates the development of gene transfer vectors that integrate genetic information effectively, safely and economically. Accordingly, significant efforts have been devoted to engineer novel tools that i) achieve high-level stable gene transfer at low toxicity to the host cell; ii) induce low levels of genotoxicity and possess a ‘safe’ integration profile with a high proportion of integrations into safe genomic locations; and iii) are associated with acceptable cost per treatment and scalable/exportable vector production to serve large numbers of patients. The Sleeping Beauty (SB) transposon has been transformed into a vector system that is fulfilling these requirements.In the CARAMBA project, we use SB transposition to genetically modify T cells with a chimeric antigen receptor (CAR) specific for the SLAMF7 antigen, that is uniformly and highly expressed on malignant plasma cells in multiple myeloma. We have demonstrated that SLAMF7 CAR-T cells confer specific and very potent anti-myeloma reactivity in pre-clinical models, and are therefore preparing a Phase I/IIa clinical trial of adoptive immunotherapy with autologous, patient-derived SLAMF7-CAR T cells in multiple myeloma (EudraCT Nr. 2019-001264-30/CARAMBA-1).Here we report on the characterization of genomic safety attributes in SLAMF7 CAR-T cells that we prepared in three clinical-grade manufacturing campaigns under good manufacturing practice (GMP), using T cells that we obtained from three healthy donor volunteers. In the SLAMF7 CAR-T cell product, we determined the average transposon copy number, the genomic insertion profile, and presence of residual SB100X transposase. The data show that the SLAMF7 CAR transposon had been inserted into the T cell genome with the close-to-random distribution pattern that is typical for SB, and with an average transposon copy number ranging between 6 and 12 per T cell. No residual SB100X transposase could be detected by Western blotting in the infusion products. With these attributes, the SLAMF7 CAR-T products satisfy criteria set forth by competent regulatory authorities in order to justify administration of SLAMF7 CAR-T cells to humans in the context of a clinical trial. These data set the stage for the CARAMBA clinical trial, that will be the first in the European Union to use virus-free SB transposition for CAR-T engineering.DisclosuresThis project is receiving funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 754658 (CARAMBA).
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- 2019
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7. A new method to monitor antigen-specific CD8+ T cells, avoiding additional target cells and the restriction to human leukocyte antigen haplotype.
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Prommersberger, S, Höfflin, S, Schuler-Thurner, B, Schuler, G, Schaft, N, and Dörrie, J
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CD8 antigen , *T cells , *HLA histocompatibility antigens , *HAPLOTYPES , *CANCER vaccines , *EPITOPES , *CANCER patients , *DENDRITIC cells - Abstract
Therapeutic vaccination of cancer patients with dendritic cells aims at inducing a strong tumor-specific T-cell response. Testing new target antigens for their immunogenicity is crucial to evaluate their suitability for this approach. Here we demonstrate a comfortable and reliable method to detect antigen-specific CD8+ T-cell responses without the knowledge of the precise T-cell epitope and without the usage of additional target cells. We used the CD8+ T cells themselves and electroporated them with RNA encoding the respective tumor antigen. The cells expressed, processed and presented the antigen and were capable of stimulating each other in functional readouts. For the model antigen MelanA, the number of interferon-γ-secreting cells obtained with this method highly correlated with the numbers obtained by exogenous peptide loading (R2=0.8). The method was also applicable for the tumor-associated antigen Wilms' tumor protein 1. This system is quick and easy to perform, independent of the donors human leukocyte antigen type and circumvents the need for additional cells as targets. It can be used in preclinical research to test new antigens for their immunogenic potential and for immunomonitoring in cancer patients. [ABSTRACT FROM AUTHOR]
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- 2015
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8. Focal pulsed field ablation in complex atrial tachycardia: First clinical experience and 1-year outcome.
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Erhard N, Englert F, Prommersberger S, Popa M, Bourier F, Reents T, Kraft H, Martinez AT, Syväri J, Tydecks M, Abdiu E, Koops E, Reiter T, Telishevska M, Lengauer S, Hessling G, Deisenhofer I, and Bahlke F
- Abstract
Background: Pulsed field ablation (PFA) has become increasingly important in the treatment of cardiac arrhythmias. In addition to single-shot devices mainly used for pulmonary vein isolation, focal PFA may provide a treatment option that increases the versatility of the technique., Objective: The purpose of this study was to provide data on feasibility, safety, and long-term outcome of focal PFA for ablation of complex atrial tachycardia (AT)., Methods: All consecutive patients (n = 34) with complex AT treated at our department between 2022 and 2023 with a focal PFA system (CENTAURI™, Galvanize Therapeutics) were included. The majority of patients (32/34) previously had undergone at least 1 radiofrequency ablation. Established contact force-sensing catheters were used for PFA application in combination with a PFA generator. Pulsed electric field trains were conducted in a R-wave triggered manner., Results: Acute procedural success was accomplished in all patients. PFA included creation of 51 linear lesions and (re)isolation of 12 pulmonary veins. Mean procedural duration was 102.7 ± 30.3 minutes, with left atrial dwell time of 75.0 ± 24.7 minutes. Mean fluoroscopy duration was 8.7 ± 5.3 minutes. No complications occurred. After mean follow-up of 340.9 ± 130.1 days, recurrence of any AT occurred in 15 patients (44.1%). During 9 reablations, 3 gaps in previously created linear lesions were detected; the majority of recurrences (n = 6) were not related to previous PFA lesion creation., Conclusion: Focal PFA of complex AT substrates was safe and efficient. Acute procedural success was 100%. After 1 year, the majority of patients were in sinus rhythm. A minority of recurrences was caused by insufficient PFA lesion creation., Competing Interests: Disclosures Dr Bahlke participates in an educational program of Boston Scientific. Dr Erhard received travel grants from Boston Scientific and Biosense Webster. All other authors have no conflicts of interest to disclose., (Copyright © 2024 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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9. Impaired FADD/BID signaling mediates cross-resistance to immunotherapy in Multiple Myeloma.
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Munawar U, Zhou X, Prommersberger S, Nerreter S, Vogt C, Steinhardt MJ, Truger M, Mersi J, Teufel E, Han S, Haertle L, Banholzer N, Eiring P, Danhof S, Navarro-Aguadero MA, Fernandez-Martin A, Ortiz-Ruiz A, Barrio S, Gallardo M, Valeri A, Castellano E, Raab P, Rudert M, Haferlach C, Sauer M, Hudecek M, Martinez-Lopez J, Waldschmidt J, Einsele H, Rasche L, and Kortüm KM
- Subjects
- Humans, Immunotherapy methods, T-Lymphocytes, Antibodies, Monoclonal therapeutic use, Receptors, Death Domain, Fas-Associated Death Domain Protein, Multiple Myeloma drug therapy
- Abstract
The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM., (© 2023. The Author(s).)
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- 2023
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10. CAR T cells targeting Aspergillus fumigatus are effective at treating invasive pulmonary aspergillosis in preclinical models.
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Seif M, Kakoschke TK, Ebel F, Bellet MM, Trinks N, Renga G, Pariano M, Romani L, Tappe B, Espie D, Donnadieu E, Hünniger K, Häder A, Sauer M, Damotte D, Alifano M, White PL, Backx M, Nerreter T, Machwirth M, Kurzai O, Prommersberger S, Einsele H, Hudecek M, and Löffler J
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- Animals, Antifungal Agents, Aspergillus fumigatus, Cytokines, Granzymes, Mice, Perforin, T-Lymphocytes, Invasive Pulmonary Aspergillosis therapy, Receptors, Chimeric Antigen
- Abstract
Aspergillus fumigatus is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to A. fumigatus are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus -specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae. T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and exerted a direct antifungal effect against A. fumigatus hyphae. In particular, CD8
+ Af-CAR T cells released perforin and granzyme B and damaged A. fumigatus hyphae. CD8+ and CD4+ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8+ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8+ Af-CAR T cells conferred greater antifungal efficacy compared to CD4+ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA.- Published
- 2022
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11. Automated, scaled, transposon-based production of CAR T cells.
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Lock D, Monjezi R, Brandes C, Bates S, Lennartz S, Teppert K, Gehrke L, Karasakalidou-Seidt R, Lukic T, Schmeer M, Schleef M, Werchau N, Eyrich M, Assenmacher M, Kaiser A, Prommersberger S, Schaser T, and Hudecek M
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- Antigens, CD19 genetics, Antigens, CD19 metabolism, Humans, Receptors, Antigen, T-Cell, T-Lymphocytes, Immunotherapy, Adoptive methods, Receptors, Chimeric Antigen
- Abstract
Background: There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition., Methods: We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR., Results: We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1-3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns., Conclusions: We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting., Competing Interests: Competing interests: DL, CB, SL, KT, NW, MA, AK and TS are employees of Miltenyi Biotec. MH is listed as an inventor on patent applications and granted patents that have been filed by the Fred Hutchinson Cancer Research Center, Seattle, WA and the University of Würzburg that are related to CAR technologies and the use of MC DNA for genetransfer into lymphocytes and that have been licensed—in part—to industry. MH is a cofounder and equity owner of T-CURX. MSchm and MSchl are listed as inventors on granted patents of PlasmidFactory that cover the use of transposons in combination with Minicircle technology for cell transfection. No competing financial interests exist for the remaining authors., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)
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- 2022
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12. A Clinical Case of COVID-19-Associated Pulmonary Aspergillosis (CAPA), Illustrating the Challenges in Diagnosis (Despite Overwhelming Mycological Evidence).
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White PL, Springer J, Wise MP, Einsele H, Löffler C, Seif M, Prommersberger S, Backx M, and Löffler J
- Abstract
The COVID-19 pandemic has resulted in large numbers of patients requiring critical care management. With the established association between severe respiratory virus infection and invasive pulmonary aspergillosis (7.6% for COVID-19-associated pulmonary aspergillosis (CAPA)), the pandemic places a significant number of patients at potential risk from secondary invasive fungal disease. We described a case of CAPA with substantial supporting mycological evidence, highlighting the need to employ strategic diagnostic algorithms and weighted definitions to improve the accuracy in diagnosing CAPA.
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- 2022
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13. Minicircles for CAR T Cell Production by Sleeping Beauty Transposition: A Technological Overview.
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Prommersberger S, Monjezi R, Shankar R, Schmeer M, Hudecek M, Ivics Z, and Schleef M
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- Genetic Vectors, Humans, Immunotherapy, Adoptive methods, Neoplasms genetics, T-Lymphocytes, Transposases genetics, Transposases metabolism, DNA Transposable Elements, Gene Transfer Techniques, Nucleic Acids
- Abstract
Development and application of chimeric antigen receptor (CAR) T cell therapy has led to a breakthrough in the treatment of hematologic malignancies. In 2017, the FDA approved the first commercialized CD19-specific CAR T cell products for treatment of patients with B-cell malignancies. This success increased the desire to broaden the availability of CAR T cells to a larger patient cohort with hematological but also solid tumors. A critical factor of CAR T cell production is the stable and efficient delivery of the CAR transgene into T cells. This gene transfer is conventionally achieved by viral vectors. However, viral gene transfer is not conducive to affordable, scalable, and timely manufacturing of CAR T cell products. Thus, there is a necessity for developing alternative nonviral engineering platforms, which are more cost-effective, less complex to handle and which provide the scalability requirement for a globally available therapy.One alternative method for engineering of T cells is the nonviral gene transfer by Sleeping Beauty (SB) transposition. Electroporation with two nucleic acids is sufficient to achieve stable CAR transfer into T cells. One of these vectors has to encode the gene of interest, which is the CAR , the second one a recombinase called SB transposase, the enzyme that catalyzes integration of the transgene into the host cell genome. As nucleic acids are easy to produce and handle SB gene transfer has the potential to provide scalability, cost-effectiveness, and feasibility for widespread use of CAR T cell therapies.Nevertheless, the electroporation of two large-size plasmid vectors into T cells leads to high T cell toxicity and low gene transfer rates and has hindered the prevalent clinical application of the SB system. To circumvent these limitations, conventional plasmid vectors can be replaced by minimal-size vectors called minicircles (MC ). MCs are DNA vectors that lack the plasmid backbone, which is relevant for propagation in bacteria, but has no function in a human cell. Thus, their size is drastically reduced compared to conventional plasmids. It has been demonstrated that MC-mediated SB CAR transposition into T cells enhances their viability and gene transfer rate enabling the production of therapeutic doses of CAR T cells. These improvements make CAR SB transposition from MC vectors a promising alternative for engineering of clinical grade CAR T cells., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2022
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14. Generation of CAR-T Cells with Sleeping Beauty Transposon Gene Transfer.
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Prommersberger S, Monjezi R, Botezatu L, Miskey C, Amberger M, Mestermann K, Hudecek M, and Ivics Z
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- Genetic Therapy methods, Genetic Vectors genetics, Humans, Immunotherapy, Adoptive methods, T-Lymphocytes, Transposases genetics, Transposases metabolism, DNA Transposable Elements, Gene Transfer Techniques, Receptors, Antigen, T-Cell metabolism, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen metabolism
- Abstract
Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be economically viable. Transposon-based gene transfer satisfies these needs, and is currently being evaluated in clinical trials. In this protocol we describe the basic Sleeping Beauty (SB) transposon vector components required for stable gene integration in human cells, with special emphasis on minicircle DNA vectors and the use of synthetic mRNA. We provide a protocol for functional validation of the vector components in cultured human cell lines on the basis of fluorescent reporter gene expression. Finally, we provide a protocol for CAR-T cell engineering and describe assays that address transgene expression, biological potency and genomic vector copy numbers in polyclonal cell populations. Because transposons allow virus-free gene transfer with naked nucleic acids, the protocol can be adopted by any laboratory equipped with biological safety level S1 facilities., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2022
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15. CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma.
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Prommersberger S, Reiser M, Beckmann J, Danhof S, Amberger M, Quade-Lyssy P, Einsele H, Hudecek M, Bonig H, and Ivics Z
- Subjects
- Genetic Therapy, Humans, Immunotherapy, Adoptive, Signaling Lymphocytic Activation Molecule Family, T-Lymphocytes, Multiple Myeloma therapy
- Abstract
Clinical development of chimeric antigen receptor (CAR)-T-cell therapy has been enabled by advances in synthetic biology, genetic engineering, clinical-grade manufacturing, and complex logistics to distribute the drug product to treatment sites. A key ambition of the CARAMBA project is to provide clinical proof-of-concept for virus-free CAR gene transfer using advanced Sleeping Beauty (SB) transposon technology. SB transposition in CAR-T engineering is attractive due to the high rate of stable CAR gene transfer enabled by optimized hyperactive SB100X transposase and transposon combinations, encoded by mRNA and minicircle DNA, respectively, as preferred vector embodiments. This approach bears the potential to facilitate and expedite vector procurement, CAR-T manufacturing and distribution, and the promise to provide a safe, effective, and economically sustainable treatment. As an exemplary and novel target for SB-based CAR-T cells, the CARAMBA consortium has selected the SLAMF7 antigen in multiple myeloma. SLAMF7 CAR-T cells confer potent and consistent anti-myeloma activity in preclinical assays in vitro and in vivo. The CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial with virus-free CAR-T cells in Europe, and the first clinical trial that uses advanced SB technology worldwide., (© 2021. The Author(s).)
- Published
- 2021
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16. Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with multiple myeloma.
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Da Vià MC, Dietrich O, Truger M, Arampatzi P, Duell J, Heidemeier A, Zhou X, Danhof S, Kraus S, Chatterjee M, Meggendorfer M, Twardziok S, Goebeler ME, Topp MS, Hudecek M, Prommersberger S, Hege K, Kaiser S, Fuhr V, Weinhold N, Rosenwald A, Erhard F, Haferlach C, Einsele H, Kortüm KM, Saliba AE, and Rasche L
- Subjects
- Aged, Antigens, Neoplasm metabolism, DNA Copy Number Variations genetics, Homozygote, Humans, Magnetic Resonance Imaging, Male, Multiple Myeloma diagnostic imaging, B-Cell Maturation Antigen genetics, Gene Deletion, Multiple Myeloma genetics, Multiple Myeloma immunology, Receptors, Chimeric Antigen immunology, T-Lymphocytes immunology
- Abstract
B cell maturation antigen (BCMA) is a target for various immunotherapies and a biomarker for tumor load in multiple myeloma (MM). We report a case of irreversible BCMA loss in a patient with MM who was enrolled in the KarMMa trial ( NCT03361748 ) and progressed after anti-BCMA CAR T cell therapy. We identified selection of a clone with homozygous deletion of TNFRSF17 (BCMA) as the underlying mechanism of immune escape. Furthermore, we found heterozygous TNFRSF17 loss or monosomy 16 in 37 out of 168 patients with MM, including 28 out of 33 patients with hyperhaploid MM who had not been previously treated with BCMA-targeting therapies, suggesting that heterozygous TNFRSF17 deletion at baseline could theoretically be a risk factor for BCMA loss after immunotherapy.
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- 2021
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17. Antibody-Based CAR T Cells Produced by Lentiviral Transduction.
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Prommersberger S, Hudecek M, and Nerreter T
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- Biomarkers, Genetic Engineering, Humans, Immunomagnetic Separation methods, Immunophenotyping, Immunotherapy, Adoptive methods, Lymphocyte Activation immunology, Receptors, Antigen, T-Cell genetics, Receptors, Chimeric Antigen genetics, Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, Genetic Vectors genetics, Lentivirus genetics, Receptors, Antigen, T-Cell immunology, Receptors, Chimeric Antigen immunology, T-Lymphocytes immunology, T-Lymphocytes metabolism, Transduction, Genetic
- Abstract
One promising approach to treat hematologic malignancies is the usage of patient-derived CAR T cells. There are continuous efforts to improve the function of these cells, to optimize their receptor, and to use them for the treatment of additional types of cancer and especially solid tumors. In this protocol, an easy and reliable approach for CAR T cell generation is described. T cells are first isolated from peripheral blood (here: leukoreduction system chambers) and afterwards activated for one day with anti-CD3/CD28 Dynabeads. The gene transfer is performed by lentiviral transduction and gene transfer rate can be verified by flowcytometric analysis. Six days after transduction, the stimulatory Dynabeads are removed. T cells are cultured in interleukin-2 conditioned medium for several days for expansion. There is an option to expand CAR T cells further by co-incubation with irradiated, antigen-expressing feeder cell lines. The CAR T cells are ready to use after 10 (without feeder cell expansion) to 24 days (with feeder cell expansion). © 2020 The Authors. Basic Protocol: Generation of CAR T cells by lentiviral transduction., (© 2020 The Authors.)
- Published
- 2020
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18. Novel targets and technologies for CAR-T cells in multiple myeloma and acute myeloid leukemia.
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Prommersberger S, Jetani H, Danhof S, Monjezi R, Nerreter T, Beckmann J, Einsele H, and Hudecek M
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- Gene Transfer Techniques, Humans, Leukemia, Myeloid, Acute immunology, Molecular Targeted Therapy methods, Molecular Targeted Therapy trends, Multiple Myeloma immunology, Oncolytic Virotherapy adverse effects, Oncolytic Viruses physiology, Receptors, Antigen, T-Cell physiology, Receptors, Antigen, T-Cell therapeutic use, Receptors, Chimeric Antigen metabolism, Signaling Lymphocytic Activation Molecule Family antagonists & inhibitors, Signaling Lymphocytic Activation Molecule Family immunology, fms-Like Tyrosine Kinase 3 antagonists & inhibitors, fms-Like Tyrosine Kinase 3 immunology, Immunotherapy, Adoptive methods, Leukemia, Myeloid, Acute therapy, Multiple Myeloma therapy, Receptors, Chimeric Antigen therapeutic use, T-Lymphocytes physiology
- Published
- 2018
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19. SLAMF7-CAR T cells eliminate myeloma and confer selective fratricide of SLAMF7 + normal lymphocytes.
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Gogishvili T, Danhof S, Prommersberger S, Rydzek J, Schreder M, Brede C, Einsele H, and Hudecek M
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- Animals, Antibodies, Monoclonal, Humanized, Heterografts, Humans, Lymphocytes immunology, Mice, Multiple Myeloma pathology, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell immunology, Signaling Lymphocytic Activation Molecule Family analysis, T-Lymphocytes immunology, T-Lymphocytes transplantation, Immunotherapy, Adoptive methods, Multiple Myeloma therapy, Signaling Lymphocytic Activation Molecule Family immunology
- Abstract
SLAMF7 is under intense investigation as a target for immunotherapy in multiple myeloma. In this study, we redirected the specificity of T cells to SLAMF7 through expression of a chimeric antigen receptor (CAR) derived from the huLuc63 antibody (elotuzumab) and demonstrate that SLAMF7-CAR T cells prepared from patients and healthy donors confer potent antimyeloma reactivity. We confirmed uniform, high-level expression of SLAMF7 on malignant plasma cells in previously untreated and in relapsed/refractory (R/R) myeloma patients who had received previous treatment with proteasome inhibitors and immunomodulatory drugs. Consequently, SLAMF7-CAR T cells conferred rapid cytolysis of previously untreated and R/R primary myeloma cells in vitro. In addition, a single administration of SLAMF7-CAR T cells led to resolution of medullary and extramedullary myeloma manifestations in a murine xenograft model in vivo. SLAMF7 is expressed on a fraction of normal lymphocytes, including subsets of natural killer (NK) cells, T cells, and B cells. After modification with the SLAMF7-CAR, both CD8
+ and CD4+ T cells rapidly acquired and maintained a SLAMF7- phenotype and could be readily expanded to therapeutically relevant cell doses. We analyzed the recognition of normal lymphocytes by SLAMF7-CAR T cells and show that they induce selective fratricide of SLAMF7+/high NK cells, CD4+ and CD8+ T cells, and B cells. Importantly, however, the fratricide conferred by SLAMF7-CAR T cells spares the SLAMF7-/low fraction in each cell subset and preserves functional lymphocytes, including virus-specific T cells. In aggregate, our data illustrate the potential use of SLAMF7-CAR T-cell therapy as an effective treatment against multiple myeloma and provide novel insights into the consequences of targeting SLAMF7 for the normal lymphocyte compartment., (© 2017 by The American Society of Hematology.)- Published
- 2017
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20. Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells.
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Hoyer S, Gerer KF, Pfeiffer IA, Prommersberger S, Höfflin S, Jaitly T, Beltrame L, Cavalieri D, Schuler G, Vera J, Schaft N, and Dörrie J
- Subjects
- Cell Differentiation, Cells, Cultured, Cytokines metabolism, Dendritic Cells transplantation, Electroporation, Gene Expression Profiling, Humans, MART-1 Antigen genetics, Microarray Analysis, Cancer Vaccines immunology, Dendritic Cells physiology, Immunotherapy, Adoptive methods, MART-1 Antigen metabolism, Monocytes physiology, RNA, Messenger genetics
- Abstract
For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs) is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs). However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs' immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1), and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.
- Published
- 2015
- Full Text
- View/download PDF
21. Generation of CD8(+) T cells expressing two additional T-cell receptors (TETARs) for personalised melanoma therapy.
- Author
-
Höfflin S, Prommersberger S, Uslu U, Schuler G, Schmidt CW, Lennerz V, Dörrie J, and Schaft N
- Subjects
- CD8-Positive T-Lymphocytes immunology, Cell Line, Tumor, Chaperonin Containing TCP-1 immunology, Humans, Immunotherapy, Adoptive, Melanoma immunology, Precision Medicine, Skin Neoplasms immunology, gp100 Melanoma Antigen immunology, CD8-Positive T-Lymphocytes metabolism, Melanoma therapy, Receptors, Antigen, T-Cell metabolism, Skin Neoplasms therapy
- Abstract
Adoptive T-cell therapy of cancer often fails due to the tumor cells' immune escape mechanisms, like antigen loss or down-regulation. To anticipate immune escape by loss of a single antigen, it would be advantageous to equip T cells with multiple specificities. To study the possible interference of 2 T-cell receptors (TCRs) in one cell, and to examine how to counteract competing effects, we generated TETARs, CD8(+) T cells expressing two additional T-cell receptors by simultaneous transient transfection with 2 TCRs using RNA electroporation. The TETARs were equipped with one TCR specific for the common melanoma antigen gp100 and one TCR recognizing a patient-specific, individual mutation of CCT6A (chaperonin containing TCP1, subunit 6A) termed "CCT6A(m) TCR." These CD8(+) T cells proved functional in cytokine secretion and lytic activity upon stimulation with each of their cognate antigens, although some reciprocal inhibition was observed. Murinisation of the CCT6A(m) TCR increased and prolonged its expression and increased the lytic capacity of the dual-specific T cells. Taken together, we generated functional, dual-specific CD8(+) T cells directed against a common melanoma-antigen and an individually mutated antigen for the use in personalised adoptive T-cell therapy of melanoma. The intended therapy would involve repetitive injections of the RNA-transfected cells to overcome the transiency of TCR expression. In case of autoimmunity-related side effects, a cessation of treatment would result in a disappearance of the introduced receptors, which increases the safety of this approach.
- Published
- 2015
- Full Text
- View/download PDF
22. Concurrent interaction of DCs with CD4(+) and CD8(+) T cells improves secondary CTL expansion: It takes three to tango.
- Author
-
Hoyer S, Prommersberger S, Pfeiffer IA, Schuler-Thurner B, Schuler G, Dörrie J, and Schaft N
- Subjects
- CD8-Positive T-Lymphocytes cytology, Cancer Vaccines immunology, Cells, Cultured, Dendritic Cells cytology, Female, Humans, Immunotherapy methods, Male, T-Lymphocytes, Helper-Inducer cytology, CD8-Positive T-Lymphocytes immunology, Cell Communication physiology, Cell Proliferation physiology, Dendritic Cells immunology, T-Lymphocytes, Helper-Inducer immunology
- Abstract
T-cell help is essential for CTL-memory formation. Nevertheless, it is unclear whether the continuous presence of CD4(+) T-helper (Th) cells is required during dendritic cell (DC)/CD8(+) T-cell encounters, or whether a DC will remember the helper signal after the Th cell has departed. This question is relevant for the design of therapeutic cancer vaccines. Therefore, we investigated how human DCs need to interact with CD4(+) T cells to mediate efficient repetitive CTL expansion in vitro. We established an autologous antigen-specific in vitro system with monocyte-derived DCs, as these are primarily used for cancer vaccination. Contrary to common belief, a sequential interaction of licensed DCs with CD8(+) T cells barely improved CTL expansion. In sharp contrast, simultaneous encounter of Th cells and CTLs with the same DC during the first in vitro encounter is a prerequisite for optimal subsequent CTL expansion in our in vitro system. These data suggest that, in contrast to DC maturation, the activation of DCs by Th cells, which is necessary for optimal CTL stimulation, is transient. This knowledge has significant implications for the design of new and more effective DC-based vaccination strategies. Furthermore, our in vitro system could be a valuable tool for preclinical immunotherapeutical studies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
- Full Text
- View/download PDF
23. [Baytinal anesthesia in geriatric surgery].
- Author
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PROMMERSBERGER S
- Subjects
- Aged, Aged, 80 and over, Humans, Anesthesia, Anesthesiology, Surgical Procedures, Operative, Thiobarbiturates
- Published
- 1959
24. [Lasonil in the varicose symptom complex and traumatic hematomas].
- Author
-
PROMMERSBERGER S and LANGER K
- Subjects
- Humans, Anticoagulants, Hematoma therapy, Heparin therapy, Hyaluronoglucosaminidase therapy, Varicose Veins therapy
- Published
- 1962
25. [On the problem of roentgen preirradiation and difficulty in histological diagnosis of osteogenic sarcoma].
- Author
-
PROMMERSBERGER S
- Subjects
- Humans, Bone Neoplasms, Neoplasms, Osteosarcoma radiotherapy, Sarcoma
- Published
- 1962
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