Detailed overview of all regimens administered as bridging therapy for each patient.

BACKGROUND:T-cell leukemias and lymphomas (TCL) are a rare and heterogeneous group of cancers with limited treatment options and an overall poor prognosis. In contrast to B-cell malignancies, the development of cellular therapies for TCL has not yet resulted in an approved product. The development of autologous CAR T cell approaches is challenging because of the shared antigens between the malignant T lymphocytes and healthy T cells, that can lead to fratricide, product contamination, manufacturing limitations, and T-cell aplasia. METHODS:To address these limitations, we developed CD4-specific chimeric antigen receptor (CAR) natural killer (NK) cells as an off-the-shelf immunotherapeutic strategy. CD4 CAR NK cells were generated from peripheral blood via retroviral transduction, and cytotoxicity was tested against 17 TCL cell lines representing six TCL subtypes. Additionally, RNA sequencing was performed to profile NK cell ligand expression across these cell lines. We further characterized CD4 CAR NK cells, cultivated in gas-permeable G-Rex culture plates, and assessed their killing capacities against primary patient-derived TCL samples. RESULTS:CD4 CAR NK cells showed CAR expression levels of up to 60% and demonstrated natural and specific cytotoxicity across T-cell malignancy cell lines. NK ligand analysis of the cell lines highlights how the balance of inhibitory versus activating signals shapes both natural and CAR-mediated responses. Using gas-permeable G-Rex culture plates, we developed a standardized manufacturing protocol, achieving over 15-fold expansion by day 14 and over 50-fold by day 21 post-isolation. Cells maintained high viability, transduction efficiency, and cytotoxic functionality, and exhibited potent activity against primary patient-derived TCL samples. CONCLUSIONS:Together, these findings demonstrate the feasibility, scalability, and therapeutic potential of CD4-directed CAR NK cells in T-cell malignancies, supporting their further development towards clinical application.

Cytokine release syndrome (CRS) is a severe immune-mediated toxicity frequently associated with immunotherapies such as CAR-T cells and bispecific T-cell engagers. Predicting CRS in humans remains difficult because current animal models and in vitro systems do not adequately capture the complex, systemic, and patient-specific nature of pathological inflammation. This perspective highlights key mechanistic and methodological limitations in current immunotoxicology approaches and proposes an immune-related Adverse Outcome Pathway (irAOP) framework adapted for biotherapeutics. The irAOP approach structures CRS as a sequence of interconnected key events (KEs), including immune cell activation, recruitment of pro-inflammatory cells, and excessive cytokine release. These KEs are harmonized from existing AOPs to distinguish pathological inflammation from normal immune responses. Emphasis is placed on downstream effects such as endothelial activation and vascular leakage, which are central to CRS severity. Clinical manifestations - including fever, hypotension, hypoxia, edema, and multi-organ dysfunction - are linked to late-stage KEs, enabling a mechanistic bridge between molecular events and patient outcomes. Insights from COVID-19-related irAOP development and international initiatives like the imSAVAR consortium inform a roadmap for improving CRS prediction. This includes defining context of use, strengthening mechanistic evidence through quantitative key event relationships, and advancing human-relevant New Approach Methodologies (NAMs). By integrating advanced in vitro and ex vivo systems within an irAOP framework, fragmented data can be transformed into coherent, decision-relevant tools. Overall, this strategy supports a shift toward predictive, human-centered approaches for assessing and managing CRS risk in immunotherapy.

SUMMARY:Adoptive cellular immunontherapies, such as chimeric antigen receptor (CAR) T cell therapy, have transformed cancer treatment, yet challenges such as resistance, relapse, and high costs limit their efficacy and accessibility. A comprehensive understanding of cellular heterogeneity and molecular profiles is essential to improve these therapies. Advanced single-cell multiomics technologies have the power to analyze the complex interactions between CAR-engineered cells, immune cells, and tumor cells. However, standardized single-cell multiomics computational pipelines specifically tailored to CAR-engineered cell products are lacking. Due to the synthetic nature of CAR transgenes, additional steps for reliable identification and characterization of CAR-positive cells are required but not included in existing data-processing workflows. To address this, we present CERTOMICS, a Nextflow-based, CAR-aware pipeline offering enhanced CERTainty in immunophenotyping and data interpretation, tailored for single-cell multiOMICSprofiling of adoptive cellular immunotherapies. The pipeline standardizes processing 10x Genomics single-cell multiomics data and integrates CAR-specific identification and quality control. Additionally, a curated repository of CAR construct sequences and annotation data is provided, serving as an extensible resource to support the analysis and development of CAR T cell therapies. AVAILABILITY AND IMPLEMENTATION:Detailed documentation of this pipeline, along with a resource on latest FDA-approved CAR therapies is available on our website: https://fraunhofer-izi.github.io/Living-Drugs-Wiki/. The data underlying this article are available on GitHub at https://github.com/fraunhofer-izi/CERTOMICS. The code is also published on Zenodo at https://doi.org/10.5281/zenodo.18709693.

Risk stratification is an important tool in clinical decision-making, yet current approaches often fail to translate sophisticated survival analysis into actionable clinical criteria. We present a novel method for training any neural network architecture on any data modality to identify prognostically distinct patient groups by directly optimizing for survival heterogeneity across patient clusters. We evaluate the method in simulation experiments and demonstrate its utility in practice by applying it to two distinct cancer types: analyzing laboratory parameters from multiple myeloma (MM) patients using the CoMMpass dataset and computed tomography images from non-small cell lung cancer (NSCLC) patients using the Lung1 dataset. Post-hoc explainability analyses uncover clinically meaningful features determining group assignments, which align well with established risk factors in both cases. Our findings in MM were externally validated using the GMMG-MM5 study dataset, while the NSCLC findings were validated with data from our own institution, thus lending strong weight to the method’s utility. This pan-cancer, model-agnostic approach enables the discovery of novel prognostic signatures across diverse data types while providing interpretable results that promise to complement treatment personalization and clinical decision-making in oncology and beyond.

To date, prediction of manufacturing failure for CAR T cells is still lacking. In a retrospective multivariate analysis of our 368 manufactured CAR-T cell batches (tisagenlecleucel), we investigated potential factors that might be associated with manufacturing failure. In this letter to the editor, we provide a summary of our findings and outline recommendations for future advanced quality control strategies.

Proportion of CAR-positive cells in T cell cultures from day 7 after CAR-T infusion used for the in vitro cytotoxicity assay.

Overview of bridging therapies and clinical characteristics.

ABSTRACT:Graft-versus-host disease (GVHD) remains one of the major complications following allogeneic hematopoietic cell transplantation. Currently, immunosuppressants are used for GVHD prophylaxis and treatment in most transplantation recipients. Due to their systemic, nonspecific mode of action, this treatment regimen is frequently associated with severe toxic side effects, opportunistic infections, as well as cancer relapse when treating hematologic malignancies. By using short-term ex vivo modulation of hematopoietic cell transplants with the anti-human CD4 antibody MAX.16H5, we have developed a novel immune tolerance-inducing strategy enabling potent GVHD prevention. Functional in vitro assays and transcriptome profiling data suggest impaired T-cell receptor signaling and a shift toward an interleukin-10-dependent regulatory phenotype as the primary mechanism of action of anti-human CD4 antibody treatment, leading to significantly reduced activation and proliferation of CD4+ and CD8+ T cells. A one-time incubation of hematopoietic transplants with MAX.16H5 prolongs survival of NSG (NOD.Cg-PrkdcSCID Il2rgtm1Wjl/SzJ) mice and reduces signs of GVHD manifestation as effectively as repeated application with clinically applied immunosuppressants, making it a safe and effective immunotherapy for GVHD prevention.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of relapsed or refractory multiple myeloma (RRMM), yet outcomes remain heterogenous. The prognostic role of body composition in this context is unknown. We retrospectively analyzed 108 RRMM patients treated with anti-B-cell maturation antigen (BCMA) CAR T-cell therapy. Pre-treatment Computed tomography imaging was utilized to quantify total adipose tissue (TAT), subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and skeletal muscle area to assess sarcopenia. Longitudinal flow cytometric and single-cell multi-omic analyses were conducted to characterize the quantitative and qualitative influences of body composition on the immune microenvironment. Patients with BMI < 25 kg/m(2) experienced significantly worse overall survival (OS) compared to high-BMI patients. Reduced TAT, primarily driven by low SAT, was associated with inferior OS, diminished response, and elevated soluble BCMA. Sarcopenia independently predicted poorer OS, while progression-free survival was unaffected by the respective parameters. Low SAT and sarcopenia correlated with lower bystander T-cell counts at leukapheresis. Longitudinal T-cell receptor sequencing and single-cell transcriptomics revealed diminished cytotoxic and interferon signaling, reduced T-cell clonality, and increased oxidative phosphorylation activity following CAR T-cell infusion. Our findings identify low SAT and sarcopenia as prognostic biomarkers that influence survival, therapeutic response, and immunometabolic profiles. Their quantification through standard imaging techniques offers a cost-effective strategy for early risk stratification and individualized management in CAR T-cell therapy.

Fig. S1: PFS analysis of the BsAb vs other bridging groups. Fig. S2: Quantification of CAR-T cells using MFC vs PCR. Fig. S3: Correlation of sBCMA levels with IMWG response criteria. Fig. S4: PFS analysis of patients with higher vs lower than median sBCMA on the day of CAR-T infusion. Fig. S5: Impact of bridging with BsAb vs other BT groups on absolute counts of T cell subsets. Fig. S6: Absolute numbers of CAR-T cells in the bridging groups. Fig. S7: Proportions of CAR-T cells in the samples used for the in vitro cytotoxicity assay per bridging group. Fig. S8: Proportions of naive and memory Tregs in the bridging groups. Fig. S9: Overview of T cell subsets stratified by response to bridging. Fig. S10: Alluvials of T cell subsets among the bridging groups. Fig. S11: Checkpoint marker expression stratified by response to bridging therapy. Fig. S12: Proportions of PD-1+CD4+ and PD-1+CD8+ non-transduced T cells amongst the bridging groups. Fig. S13: Overview of sample numbers in the bridging groups included for single-cell multi-omics sequencing. Fig. S14: Numbers of circulating plasma cells.

Fig. S15: Gating scheme used for quantification of CAR-T cells. Fig. S16: Gating scheme used for immune checkpoint marker detection on non-transduced and CAR-T cells. Fig. S17: Gating scheme used for T cell subsets. Fig. S18: Gating scheme used for detection of perforin and granzyme B on non-transduced and CAR-T cells. Fig. S19: Gating scheme used for flow cytometry readout of the in vitro cytotoxicity assay.

Background:Clinical decision-making for patients with community-acquired pneumonia (CAP) at risk of organ dysfunction and death is currently guided by clinical evaluation and scores. Every fifth patient with CAP requires admission to the ICU, with a subsequent high mortality; delayed admission to the ICU increases this risk. Research Question:Can a transcriptomic signature improve identification of at-risk patients compared with conventional scores and metrics? Study Design and Methods:Time-course transcriptomic data were obtained from blood samples taken from 455 participants in 41 centers enrolled in the Progression of Community-Acquired Pneumonia in the Hospital (PROGRESS) trial, a prospective observational cohort study of hospitalized patients with CAP who did not initially require organ support. Discovery (n = 240) and validation (n = 215) cohorts were randomly assigned. Transcriptome data were analyzed for association with a severe CAP course, defined as a composite of requirement for ICU admission or 28-day mortality. Predictive performance of the gene expression profiles was compared against clinical scores and serum markers, and validated in publicly available transcriptomic data sets. Results:A 5-gene signature consisting of SIGLEC14, TNFSF14, YOD1, CLEC4A, and KLRB1 (STYCK) was identified and validated to predict clinical deterioration with subsequent ICU admission or 28-day mortality (AUCdiscovery, 0.82; 95% CI, 0.71-0.90; P = 0.00000065; AUCvalidation, 0.81; 95% CI, 0.70-0.90; P = 0.0000013). The signature outperformed clinical scores in predicting severe CAP and improved prediction when added to the Sequential Organ Failure Assessment score (AUCSOFA, 0.70 vs AUCSOFA+STYCK, 0.83; P = .0002). Prognostic value was confirmed in 6 of 17 publicly available sepsis cohorts, particularly those with low case fatality. Interpretation:We identified a 5-gene transcriptomic signature that, taken soon after hospital admission, was shown to predict disease course of hospitalized patients with CAP. STYCK was superior to conventional, currently used scores and clinical metrics in predicting this deterioration and improved, if added to the Sequential Organ Failure Assessment, its performance. Clinical Trial Registration:ClinicalTrials.gov; No.: NCT02782013; URL: www.clinicaltrials.gov.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by immense heterogeneity, not only between patients but also within malignant populations, leading to limited curative outcomes. These challenges underscore the urgent need for innovative therapies, such as chimeric antigen receptor (CAR)-T cell therapies, which show promising antileukemic activity but remain limited by toxicity and antigen escape. In contrast, CAR-Natural Killer (NK) cell therapies offer an improved safety profile, off-the-shelf availability, and high inherent cytolytic activity. Owing to the heterogeneous nature of AML, multiple target antigens must be addressed and validated. However, there have been no side-by-side comparisons of different AML antigens targeted by the same CAR construct for NK cell-based therapy. This study addressed CD33- and CD123-targeting CAR-NK cells, both in vitro and in vivo, in a fully matched experimental setting. Functional analyses revealed a significant increase in killing efficacy of both engineered CAR-NK cell products, compared to non-transduced NK cells. Additionally, in-depth transcriptomic analyses revealed the upregulation of motility and cytotoxicity pathways in CD123-CAR-NK cells compared to CD33-CAR-NK cells. Consistent with this, only CD123-targeting CAR-NK cells increased survival in a xenograft AML model and exhibited significant effects against primary AML in vitro and in vivo. Furthermore, upscaling of CD123-CAR-NK cell manufacturing using the CliniMACS Prodigy System, resulted in highly functional CAR-NK cell preparations. Together with the transcriptional and functional profiling, this work represents an encouraging proof-of-concept for the application of genetically engineered CD123-targeting CAR-NK cell preparations in advanced AML therapy.

Chimeric antigen receptor (CAR) T cells and bispecific T cell engagers have become integral components in the treatment of relapsed/refractory multiple myeloma. We report a 63-year-old male who received ciltacabtagene autoleucel CAR T cells and the GPRC5D × CD3 bispecific talquetamab for early relapse of his multiple myeloma. Nine months after CAR T therapy, he developed a symptomatic leukemic peripheral T cell lymphoma with cutaneous and intestinal involvement. Longitudinal single-cell RNA and T cell receptor sequencing of peripheral blood and bone marrow revealed two hyperexpanded CAR-carrying T cell clones. These expanded clones exhibited an exhausted effector-memory T cell transcriptional signature, and the neoplasm itself was sensitive to dexamethasone treatment. The immunophenotypic and transcriptional alterations of these abnormal T cells resembled those of T-large granular lymphocytic leukemia. Spatial transcriptomes of skin lesions confirmed the aberrant CAR-expressing T cells. Whole-genome sequencing revealed three distinct integration sites, within the introns of ZGPAT, KPNA4 and polycomb-associated noncoding RNAs. Before and after CAR T whole-genome analyses implicated clonal outgrowth of a TET2-mutated precursor propelled by additional subclone-specific loss of heterozygosity and other secondary mechanisms. This case highlights the evolution of a CAR-carrying peripheral T cell lymphoma following CAR T cell and bispecific T cell engager therapy, offering critical insights into the clonal evolution from a predisposed hematopoietic precursor to a mature neoplasm.

Hematotoxicity and infections are the main drivers of non-relapse mortality after chimeric antigen receptor (CAR)-T therapy. Consequently, reliable predictive biomarkers are highly needed to improve risk assessment and optimize patient management. In this study, we applied the immune-related adverse outcome pathway concept to delineate key events and risk factors of CAR-T-associated hematotoxicity. To identify predictive biomarkers, we performed flow cytometry and multiplex assays before and early after CAR-T infusion on 78 patients (ide-cel n = 31; axi-cel n = 24; and cilta-cel n = 23) undergoing CAR-T therapy. Severe hematotoxicity was linked to endothelial dysfunction, as evidenced by reduced levels of ANG1, soluble selectins, and increased soluble VCAM-1 (sVCAM-1) early after CAR-T infusion. Increased sVCAM-1, reflecting endothelial dysfunction, elevated soluble IL-2R (sIL-2R), indicating a proinflammatory state, and high tumor burden before lymphodepletion were key risk factors for CAR-T-associated hematotoxicity. Patients with elevated sVCAM-1 and sIL-2R at baseline (pre-lymphodepletion) exhibited significantly reduced overall survival (OS) (sVCAM-1; P = 0.0009), prolonged Grade 4 neutropenia (sVCAM-1; 12.1 vs. 6.0 days; P = 0.0016), more aplastic neutrophil recovery (5% vs. 30%; P = 0.007), and more severe infections (22.4% vs. 55%; P = 0.011). Baseline sIL-2R and sVCAM-1 demonstrated robust predictive value for prolonged neutropenia, severe infections, and mortality independently of key clinical variables such as the underlying disease and CAR-T product. Integration of these markers improves existing models and can help to refine risk assessment and guide individualized patient management in CAR-T therapy.

Vascular leakage (VL) is a severe pathology occurring in a broad range of scenarios, e.g., during sepsis, cytokine storms, or as side effect of immunotherapies. Its severity is underlined by the high lethality rate of 20-30% for the systemic capillary leakage syndrome. While many compounds are reported to affect endothelial cell (EC)-activation, exact mechanisms behind VL remain unclear. We analyzed activation, viability, cytokine secretion, and relative permeability of human umbilical vein endothelial cells (HUVECs) upon treatment with 16 different stimuli. Relative HUVEC-permeability was assessed in a trans-well-based leakage assay in presence or absence of human peripheral blood mononuclear cells (PBMCs). HUVEC-activation is characterized by correlating upregulation of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin, as well as production of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, and IL-6. Strong HUVEC-activation and reduced viability was observed upon treatment with IL-1β, tumor necrosis factor (TNF)-α, a TGN1412-induced cytokine cocktail (SNTGN1412), thrombin, and lipopolysaccharide (LPS). Only thrombin, SNTGN1412, and vascular endothelial growth factor (VEGF) led to increased relative permeability, while other compounds associated with vascular leakage, including TNF-α, IL-1β, or LPS, had no direct effect on relative HUVEC-permeability. Interestingly, co-cultures with PBMCs mediated IL-1β- and LPS- but not TNF-α-induced relative HUVEC-permeability. In our study, we show that HUVEC activation upon direct stimulation does not necessarily result in increased relative permeability or massive cytokine production. Interestingly, we could demonstrate that activated HUVECs respond with a conserved pattern of markers, independent from the stimulus used. Moreover, we observed that the interplay with immune cells is critical to enhance relative HUVEC-permeability, which however depended on the stimulus applied suggesting different mechanisms of immune-mediated VL. A better understanding of VL will uncover potential treatment-targets for patients suffering from VL and help to improve safety-assessment of leakage-associated immunotherapies.

IgG4-related disease (IgG4-RD) is a fibroinflammatory disorder in which IgG4+ B cells and T cells interact to drive chronic organ inflammation. Patients with multiorgan involvement may become refractory to standard therapy, resulting in progressive organ damage and risk of organ failure. Here, we report a patient with treatment-refractory multiorgan IgG4-RD treated with CD19-directed chimeric antigen receptor (CAR) T cells and describe the clinical and immunologic effects over more than 12 months of follow-up. A 60-year-old man with IgG4-RD involving the pancreas, hepatobiliary tract, and lungs, who was refractory to long-term immunosuppression, received autologous CD19-directed CAR T cells. Disease course was monitored longitudinally and multimodal immune profiling was performed on peripheral blood. CAR T cell therapy was well tolerated, with only grade 1 cytokine release syndrome, no neurotoxicity, and transient cytopenias without infections. Treatment induced B-cell aplasia lasting 6 months, while serum IgG4 levels normalized by month 8 and remained within the reference range thereafter. FAPI (fibroblast activation protein inhibitor) PET/CT demonstrated regression of fibroinflammatory activity, accompanied by improved lung function and quality of life. Immunosuppressive therapy was completely discontinued without disease flares for more than 12 months. B cell reconstitution consisted predominantly of naïve and transitional subsets. This was paralleled by a decline in T follicular helper cells and CD4+ cytotoxic T cells and attenuation of fibro-inflammatory and B cell-mediated signaling networks on interactome analyses. In this treatment-refractory case of multiorgan IgG4-RD, CD19-directed CAR T cell therapy induced durable, treatment-free remission with normalization of serum IgG4 and improvement across multiple clinical endpoints. Multimodal immune profiling indicates that CAR T cells can reset the B cell compartment and dampen pathogenic T cell and stromal interactions, supporting prospective evaluation of CAR T cell therapy in IgG4-RD.