57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

PD Dr. Dr.

PD Dr.

Prof. Dr.

Immunotherapies beyond cancer: repurposing sibrotuzumab and NK-cells as a synergistic approach to treat fibrosis

Immunotherapy and Gene Therapy

Immuntherapien außerhalb der Onkologie: Repurposing von Sibrotuzumab und NK-Zellen als synergistischer Ansatz zur Behandlung von Fibrose

Fibrosis occurs in almost all chronic (inflammatory) diseases such as heart failure, (pulmonary) hypertension, systemic sclerosis, fatty liver disease and graft-versus-host disease. The uncontrolled secretion of extracellular matrix and inflammatory cytokines by dysregulated fibroblasts leads to a gradual loss of tissue function and contributes to 45% of deaths in the western world. Despite a clear medical need, effective antifibrotic therapies are lacking. However, fibroblast activation protein alpha (FAPα) has emerged as a potential fibroblast-specific target. Here, we present a novel strategy using the therapeutic FAPα antibody sibrotuzumab in combination with high affinity Fc receptor expressing NK92 cells (FCR-NK92) to eliminate FAPα+ fibroblasts and treat fibrosis.In the present study, human cardiac (HAF-SRK01), pulmonary (MRC5) and dermal (HFF1) fibroblasts and FCR-NK92 ± sibrotuzumab were used in flow cytometry, cytotoxicity and western blot experiments to assess the antifibrotic potential of NK-mediated antibody-dependent cellular cytotoxicity.First, we demonstrated specific activation of sibrotuzumab-loaded FCR-NK92 in the presence of human recombinant FAPα as determined by CD107A expression in a flowcytometric degranulation assay. Specific fibroblast killing was determined via europium release assay indicating significant fibroblast lysis after 2h. As therapeutic interventions targeting fibroblasts should be cell-specific to avoid off-target effects, we used a heterocellular culture model (fibroblasts and NK-resistant non-fibroblasts) to characterize the extent of specific fibroblast killing. After 4h, the FAPα+ fibroblast population was reduced by approximately 50% in the sibrotuzumab-treated cultures, whereas the non-fibroblast population (FAPα-) remained unchanged. To evaluate a functional antifibrotic effect, we measured protein expression of collagen 1 by western blot. After overnight co-culture, collagen 1 expression was abolished in all samples treated with FCR-NK92 and sibrotuzumab compared to control.As of now, antifibrotic cellular immunotherapies remain a largely untapped therapeutic opportunity. The results of our study highlight the promising potential of targeted cellular immunotherapy for the treatment of fibrosis, offering a compelling rationale for further exploration of this approach in the management of debilitating fibrotic diseases.The authors declare no conflict of interest.

Poster

Development of a CAR T-cell based agranulocytosis mouse model

Entwicklung eines CAR T-Zell basierten Agranulozytosemausmodells

Agranulocytosis, the most severe form of granulocytopenia, is characterized by a complete loss of granulocytes in the blood. Mouse models of chronic agranulocytosis are lacking, yet such a model could provide useful insights into the regulation of neutrophil and stem cell homeostasis as well as support infectious disease and antibiotic testing. We postulate that ablation of neutrophils with anti-granulocyte CAR T-cells might be able to generate a long-lasting agranulocytosis mouse model.To prevent immunization, a murinized second generation CAR‑backbone was complemented with a scFv derived from the Gr1 (granulocyte differentiating antigen 1) hybridoma and expressed on murine T‑cells by retroviral transduction. In addition to Ly6G (lymphocyte antigen 6 complex locus) Gr1 also binds Ly6C, albeit with lower affinity. The human cell line MDA-MB‑468 was engineered to express either Ly6G or Ly6C to function as target cells. Functionality was assessed by a FACS‑based killing assay. CAR T-cells were further analyzed for their phenotype and exhaustion markers during culture. C57BL/6 mice were lymphodepleted with cyclophosphamide one day prior to CAR T-cell injection and peripheral blood granulocytes were monitored for 4 weeks.CAR T-cells showed specific in vitro killing of Ly6G expressing target cells compared to control T-cells and parental MDA‑MB‑468 target cells. As murine T-cells lowly express Ly6C, some fratricide was observed during CAR T culture. Independent of CAR expression, T‑cells change from naïve to central memory/ effector phenotype in culture. Directly after T-cell separation a transient upregulation of several exhaustion markers was observed, which regresses by day 5. Moreover, TIM-3, CTLA-4 and LAG3 indicate exhaustion. In vivo data revealed delayed regeneration of WBCs, especially monocytes, after application of CAR T-cells compared to the control group, but, likely due to loss of CAR-T, at the current conditions chronic ablation was not yet achieved.We have generated a highly functional murine CAR-construct that specifically kills Ly6G expressing cells. With the ongoing mouse experiments, we aim to establish a model of long-term immune neutropenia via Gr1 targeting CAR T‑cells. The model will address the role of neutrophils in immature hematopoiesis in vivo and will investigate CAR T longevity, anergy and exhaustion, as well as drugs in neutropenic infection models.The authors declare no conflict of interest

Implementation of a decentralized CAR-T cell manufacturing in an Austrian University hospital

Implementierung einer dezentralisierten CAR-T Zellherstellung in einer Universitätsklinik in Österreich

The development of CAR-T cell preparations has revolutionized the treatment of plasma and B-cell malignancies. However, the high price limits the supply of approved CAR-T cell products to all patients. Another goal is clinical testing of new indications and innovative approaches such as the combination therapies. In-house production of CAR-T cells has been established for this purpose.All process steps for generating CAR-T cell preparations from leukapheresis products are carried out on the CliniMACSProdigy platform in a functionally closed sterile disposable set. After 12 days, the final product is formulated after cell harvest. Transduction to CD19-specific CAR-T cells is achieved using a lentiviral expression vector (Lentigen Technology, Inc.). The quality of intermediate and end products is tested using validated methods for the following parameters: Identity, content, viability, purity, transduction efficiency (FACS), viral copy number (VCN, qPCR) and freedom from microbial growth (blood culture device), endotoxins (turbidimetric) and mycoplasma (qPCR). Compliance with predefined target ranges yields final products suitable for release.The pharmaceutical quality system of the dept. of transfusion medicine was adapted to the regulatory requirements and extended to the new equipment and methods: qualifying instruments, extensive staff training and method validation. Except for the endotoxin assay, all tests were carried out by QC. Due to the identity with the IMP in an ongoing trial preclinical testing was not required. The production was successfully established in 6 production runs (1 without transduction) and in 4 aseptic process simulations. A median of 5.2*10e9 CD3+T cells and 2.2*10e9 CAR-T cells were obtained from 10e8 CD3+T cells seeded. The viability was 97.2%, the percentage of CD3+T cells 99.7%, the transduction efficiency 44.2%, the VCN 2.77. Absence of microbial growth, mycoplasma and endotoxins resulted in releasable products. Storage tests at 2-6°C proved product stability over 48 hours.The consistent output of products ready for release demonstrates the successful implementation of an in-house CAR-T cell manufacturing. This enables clinical trials on innovative therapies and thus optimum patient care.I hereby certify that, to the best of my knowledge, no aspect of my current personal or professional circumstance places me in the position of having a conflict of interest with this presentation.

Prof. Dr. med.

Gene expression analysis and whole exome sequencing revealed the PTPN11/CEBPα/BCL2 axis as novel therapeutic vulnerability in an aggressive human MLL-ENL pre-leukemia model

Genexpressionsanalysen und Exom-Sequenzierungen zeigen die PTPN11/CEBPα/BCL2-Achse als neue therapeutische Schwachstelle in einem aggressiven menschlichen MLL-ENL-Prä-Leukämiemodell

Chromosomal rearrangements of the MLL gene (MLL-r) are strong drivers of acute leukemia with poor prognosis. During the last years, the first MLL-r targeting compounds have been developed. However, resistance has been described already. Here, we identified a highly upregulated core gene set and a secondary driver mutation in human long-term cultured CD34+ progenitors expressing the t(11;19) MLL-ENL (ME) fusion, with the intention to uncover druggable vulnerabilities in ME driven transformation.ME was introduced into human CD34+ progenitors from healthy donors via retroviral transduction. Upon ex vivo expansion and outgrowth of ME monocytic progenitors, gene expression profiling was performed via the Nanostring technology, bulk and single cell (sc) RNA sequencing. We compiled a top 30 list of ME upregulated genes, including CEBPα and its target gene BCL2. To investigate cell biological consequences of CEBPα and BCL2 disruption, genetic downregulation via lentiviral shRNA and pharmacological inhibition with small molecule compounds were explored. For genomic survey whole exome sequencing was performed revealing mutated PTPN11, an activator of CEBPα gene expression, as additional driver for ME triggered cell growth.Gene arrays identified high CEBPα levels in ME progenitors. Of note, CEBPα shRNA blocked proliferation indicating cooperativity with ME. Indeed, combination of the MLL inhibitor MI-503 and the CEBPα complex inhibitor OICR-9429 strongly reduced proliferation. Moreover, BCL2 was downregulated upon CEBPα knockdown. Hence, treatment with Venetoclax (VEN) significantly reduced growth. Combining VEN with either MI-503 or OICR-9429 further increased growth-inhibition. Additionally, exome sequencing revealed a monoclonal PTPN11(Y62S) mutation, indicating a pro-proliferative role of PTPN11. Accordingly, scRNA-seq discovered co-expression of PTPN11, CEBPA and BCL2 in ME blasts. Of note, a clinical PTPN11 inhibitor dose dependently blocked growth.Our proposed t(11;19) pre-leukemia model proves the usefulness of human CD34+ progenitors derived from healthy donors for disease modeling and drug target validation. The study sheds light into the oncogenic mechanisms of ME fusion protein activity in human CD34+ progenitors suggesting oncogenic cooperativity of ME with the PTPN11/CEBPα/BCL2 network in AML transformation. The here identified vulnerabilities might be attractive drug targets to inhibit the aggressive tumor promoting effects of ME.The authors declare no conflict of interest.

Plasma transfusion from blood donors with high neutralizyting capacity against human cytomegalovirus (HCMV) increases the neutralization capacity against HCMV in hematopoietic stem cell recipients

Plasmatransfusion von Spendern mit hoher Neutralisationskapazität gegen humanes Zytomegalievirus (HCMV) erhöht bei stammzelltransplantierten Patienten die Neutralisationskapazität gegen HCMV

Immunoglobulins have been repeatedly applied to treat human cytomegalovirus (HCMV) infections, but without clear evidence of clinical efficacy. One explanation for these disappointing results is the limited neutralizing potency of the immunoglobulins used. An increase in the neutralizing antibody titers of the treated patients has also not yet been demonstrated.In a Phase I clinical trial, we tested whether the transfer of plasma from previously identified blood donors who neutralize HCMV exceptionally well (HCMV elite neutralizers) can increase the neutralization capacity in the serum of treated patients.The study included 12 stem cell transplant recipients who were HCMV seronegative and thus at risk for HCMV reactivation and disease. Patients received one or two plasma units of elite neutralizers in each of four treatment weeks. Serum samples were collected from patients before and after treatment and on days 2 and 4 of the treatment week. As a primary endpoint, neutralization titers in patients' serum samples were determined with an immunofluorescence-based assay using HCMV strain TB40/E, resulting in time curves of neutralization titers over the entire treatment period. Correlation analyses were performed to investigate whether the increase achieved by the treatment correlates with the neutralization capacity of the transferred plasma units. To analyze strain-specific differences, samples from before and after treatment were re-evaluated with seven laboratory strains representing almost all glycoprotein genotypes.On average, the transfused plasma units had an approximately 10-fold higher mean neutralizing capacity than the patients' sera. The combined curves of all patients showed that the plasma transfusion increased the neutralization capacity of the patients, on average by 83% immediately after transfusion and by 33% after one week. Over a period of four weeks, the cumulative increase in neutralization titer was 132%. The increase achieved in individual patients was highly variable and correlated with low baseline titers of the patients and high titers of the transferred plasma.Adoptive transfer of anti-HCMV antibodies can increase neutralization titers in a relevant patient group. Patients with a low neutralization capacity may benefit from the transfer of neutralizing antibodies, whereas patients with a high neutralization capacity will probably not.None

Student

Danicopan as a Novel Therapy for Paroxysmal Nocturnal Hemoglobinuria: A Pooled Meta-Analysis

Danicopan als neuartige Therapie für die paroxysmale nächtliche Hämoglobinurie: Eine gepoolte Metaanalyse

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease characterized by complement-mediated intravascular hemolysis. Current treatments like Eculizumab and Ravulizumab have limitations, with many patients still requiring transfusions. Danicopan is a first-in-class oral small molecule drug that inhibits the complement alternative pathway component D. The drug has shown efficacy in suppressing intravascular hemolysis and controlling C3-mediated extravascular hemolysis, which may reduce the side effects of conventional treatments. This study aimed to investigate the safety and efficacy of Danicopan, a newly FDA-approved add-on treatment for PNH.We systematically searched five electronic databases to ensure comprehensive coverage: Epistemonikos, Web of Science, Medline, Scopus, and ClinicalTrials. Meta-analysis was conducted through the "R" language with the "meta" statistical package.Four multicenter trials involving 79 patients were included. Danicopan treatment significantly improved hemoglobin levels (SMD 1.536, 95% CI: 0.683 to 2.390, p &lt; 0.001) and decreased reticulocyte counts (SMD -0.996, 95% CI: -1.352 to -0.641, p &lt; 0.001). However, LDH levels did not significantly change after treatment. There was a significant increase in GPI-deficient erythrocytes but not in granulocytes. Both total and direct bilirubin levels showed significant differences from baseline to week 12 after treatment. Additionally, there was a notable improvement in FACIT scores from baseline.Our systematic review and meta-analysis support the potential of Danicopan as a viable therapeutic option for PNH patients. The targeted inhibition of factor D within the complement system by Danicopan demonstrates both safety and efficacy in managing PNH, as evidenced by our findings.Ethics Approval: Not applicable.Funding information: None.Conflict of Interest Statement: The authors declare no competing interests.Data availability Statement: The data supporting this study"s findings are available from the corresponding author upon reasonable request.Author contributions: Study concept and design: Emir Muvaffak, Muhammed Edib Mokresh; screening: Emir Muvaffak, Muhammed Edib Mokresh, Abdullah Varda; acquisition of data: Emir Muvaffak, Muhammed Edib Mokresh, Abdullah Varda, Mahmoud Lakmoush; analysis and interpretation of data: Muhammed Edib Mokresh, Merve Kabasakal Ilter; drafting of the manuscripts: All authors participated; critical revision of the manuscript: All authors participated.

Prof. Dr. rer. nat.

Challenges of scalable production and effective purification of mesenchymal stromal/stem cell derived extracellular vesicles for clinical application

Herausforderungen der skalierbaren Produktion und effektiven Reinigung von extrazellulären Vesikeln aus mesenchymalen Stroma-/Stammzellen für die klinische Anwendung

The clinical potential of extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) necessitates overcoming formidable production and purification obstacles. Ensuring a smooth transition from research to clinical implementation entails standardized production and optimization, as well as specific medium requirements. Our aim is to identify a low protein content MSC medium that is animal-component-free and lacks primary human components, but still preserves the therapeutic function of obtained EV products.The establishment of clonally immortalized MSCs (ciMSCs) represents a significant advancement that opens up new avenues for standardized processes and products. In terms of identifying a suitable MSC medium, the growth and EV secretion performance of ciMSCs was performed in different cell culture media, including DMEM media supplemented with differing concentrations of human platelet lysate (hPL) as wells as commercially serum-/xeno-free or chemically defined media. Expansion in hPL-free and chemically defined media turned out to require higher seeding densities, particular detachment agents, and plastic wares with specialized surfaces.ciMSCs could be expanded in DMEM supplemented with hPL down to 1%. However, compared to DMEM supplemented with 2.5% hPL, the population doubling time was clearly increased. The ciMSCs also exhibited consistent expandability in serum-/xeno-free and in chemically defined media. However, preliminary results imply that in hPL-reduced/-free media significantly less EVs are secreted than in regular media.Despite the reduced ciMSC-EV secretion, the therapeutic potency of resulting EVs remains to be investigated. After defining an appropriate MSC medium, future work will focus on refining downstream processing procedures for MSC-EV production, aligning with Good Manufacturing Practice standards, to facilitate the clinical application of MSC-EVs.BG is a scientific advisory board member of Innovex Therapeutics SL, Mursla Ltd, ReNeuron Ltd. and PL BioScience. He is a founding director of Exosla Ltd. All other authors declare that they have no conflicts of interest.

Ex vivo expansion of human T lymphocytes results in regulation of long noncoding RNAs (lncRNAs), which are in part associated with therapeutically relevant functions

Regulierung von long noncoding RNAs (lncRNAs) während der ex vivo Expansion humaner T Lymphozyten in Kultur

Long noncoding RNAs (lncRNAs) are RNAs with lengths exceeding 200 nucleotides without encoding proteins. Whereas noncoding elements located in intergenic regions including lncRNA were in the past largely considered as junk sequences, more recently specific functions have been ascribed to them. We aimed to characterize lncRNAs which are expressed in human T lymphocytes and their regulation during ex vivo culture.Human CD3+ lymphocytes were isolated using Fab-fragment based affinity column separation from peripheral blood of 8 male healthy donors. T cells were cultured in interleukin-2 and anti-CD3/CD28 for up to 7 days. RNA was extracted at time points T1 (freshly isolated), T2 (4h), T3 (12h), T4 (24h), T5 (3d) and T6 (7d), reverse transcribed and subjected to transcriptome analysis using Illumina TruSeq from cDNA libraries. Differential gene expression analysis was performed targeting lincRNA gene sets.The number of differentially expressed LncRNAs at 4 hr, 12 hr, 24 hr, day 3 and day 7 compared to freshly isolated T cells was 168, 193, 239, 386 and 296, respectively. Of the main upregulated RNAs in cultured T cells, lncRNAs associated with suppressing cytokine production (e.g. IL-21-AS1; IL12A-AS1), with stimulating regulatory T cell differentiation (MIR155HG) and with T cell exhaustion (FOXP4-AS1) were found. Among the most downregulated were lncRNAs associated with stimulating T cell differentiation (GATA2-AS1), inhibiting interferon gamma production (IFNG-AS1) and inhibiting immune synapse (ITGB2-AS1), pointing at a role of lncRNAs towards activation of T cells during expansion culture.Our results demonstrate regulation of lncRNA expression in T lymphocytes during ex vivo manipulation. First insights into the function of lncRNAs point to an association with initiating T cell activation during culture. A more profound knowledge on lncRNA functions and its application to T cell expansion culture might provide rationales to interfere with T cell fate during expansion culture, e.g. inhibiting senescence and promoting a more self-renewing phenotype.Es wurde kein Interessenskonflikt offengelegt.

Prof.

Gene-specific knockouts to improve the proliferation and persistence of NK cells in immunotherapy

Spezifische Gene knockouts zur Verbesserung der Proliferation und Persistenz von NK Zellen in der Immuntherapie

Natural killer (NK) cells are potent cytotoxic innate lymphocytes, and the potential of NK cell therapy to treat cancer is currently explored by many clinical trials. However, one of the main drawbacks of such approaches is the limited in vitro expansion capacity of NK cells and, upon transfusion, their insufficient in vivo persistence.We performed RNA-Seq analysis on NK cells expanded in vitro to determine gene expression changes driven by IL2 and IL15. CRISPR/Cas9-mediated genome editing was used to knockout genes negatively controlling the proliferation and restricting the expansion potential of NK cells.Primary NK cells were expanded by a factor 500 in vitro using standard pre-clinical protocols. We identified that IL2 and IL15 signaling in these cells involved the induction of negative regulators such as SOCS family genes to limit the proliferation. While meaningful for regular immune reactions, the therapeutic potential of NK cells could be improved by the elimination of inhibitory factors. Thus, we efficiently knocked out CISH, SOCS1 and SOCS2 as shown on the genomic as well as on the protein level. Indeed, these knockout cells had a competitive advantage over unmodified NK cells.Genome editing of NK cells can be used to increase the proliferative capacity of NK cells. This technical advance will be extended and allow the derivation of NK cells with strongly improved persistence and efficacy in adoptive immunotherapy.no disclosures

Advanced quality control through single EV analysis: Fingerprinting of MSC-EV populations with unique capabilities for end-product and in-process control

Erweiterte Qualitätskontrolle durch Einzel-EV-Analyse: Fingerprinting von MSC-EV-Populationen mit einzigartigen Möglichkeiten zur Endprodukt- und In-Prozess-Kontrolle

In the evolving field of Mesenchymal Stromal Cell-derived Extracellular Vesicles (MSC-EVs), ensuring consistent quality and therapeutic efficacy remains a challenge due to the complexity of EV biology and production variability. Our research advances the field by leveraging Imaging Flow Cytometry (IFCM) for an unprecedented level of quality control in MSC-EV production. This novel approach allows the direct analysis of EVs in conditioned media, bypassing the need for complex isolation steps, thus enabling real-time, in-process monitoring.Central to our innovation is the detailed analysis of EV subpopulations using both, ubiquitous EV markers (CD9, CD63, CD81) and those specific to the MSC or human platelet lysate (hPL) environment (CD41, CD61, CD235a), alongside MSC-specific markers (CD90, CD105). This dual-focus analysis permits the development of a detailed 'fingerprint' for MSC-EVs, facilitating the identification of batch inconsistencies and the evaluation of hPL quality.By comparing these 'fingerprints,' our method not only ensures batch-to-batch consistency but also opens new avenues for monitoring and enhancing the quality of MSC-EV production.This approach represents a significant step forward in the standardization of MSC-EV therapies, potentially accelerating their transition from bench to bedside and highlighting the role of our method in advancing personalized and regenerative medicine.BG is a member of a scientific advisory board of Innovex Theraoeutics SL, Mursla Ltd, ReNeuron Ltd and PL BIoscience. He is a founding director of Exosla Ltd. All other authors declare no conflicts of interest.

Advanced Quality Control Through Single EV Analysis: Fingerprinting of MSC-EV Populations with Unique Capabilities for End-Product and In-Process Control

Posterausstellung 5
Immuntherapy and Gene Therapy

Appointment

Programme

Posterausstellung 5Immuntherapy and Gene Therapy

Appointment

Programme

Posterausstellung 5
Immuntherapy and Gene Therapy