Background & Aims Translation of cell- and gene-based therapies, including CAR T cells, from academic research to market approval remains challenging due to scale-up, manufacturing, regulatory, and clinical integration hurdles. To assess whether production capacity contributes to this gap, we mapped current practices, technologies, organizational structures, and challenges within ATMP development and manufacturing facilities across Europe. Methodology A survey was distributed to academic, non-profit, and commercial ATMP facilities affiliated with EBMT, NXTGEN Hightech, DARE-NL, T2EVOLVE, and the GoCART Coalition to collect data on their activities, technologies and organization. Responses were analyzed to identify trends, variability in approaches, and challenges. Results Responses (n=92) were received from 33 countries (Europe n=75), mainly from the Netherlands (n=18), Spain (n=13), Italy (n=7), and Germany (n=7). Most respondents were academic centers (90%), primarily affiliated with EBMT (66%), treating adult (47%), pediatric (13%), or both patient (40%) with ATMPs. ATMP Facilities mainly operate as stand-alone units or are embedded within clinical or pharmaceutical departments, with ∼75% sharing resources with host organizations.Leukapheresis (n=35/44), bone marrow (n=13), and umbilical cord tissue (n=9) were the most common cell source Frequently used cell types included stem cells (n=20), αβ T cells (n=18), MSCs (n=14), and NK cells (n=13). Most centers developed CTMPs (n=42), GTMPs (n=29) and TEPs (n=9). Gene delivery relied mainly on viral vectors (n=22) and electroporation (n=14). Genetic modification approaches included viral vectors, transposons, CRISPR/Cas KO (n=6), and KI (n=5). Nearly half of facilities used the CliniMACS Prodigy, valued for its closed-system design, though high costs, limited scalability, process inflexibility, and training demands were key barriers.ATMP production area correlated with cleanroom numbers. Personnel growth of 20-50% and production increases of 20-100% (academic) and 20-300% (private) are projected. Key challenges include financial constraints, regulatory complexity, GMP readiness, and scalable processes. Conclusion This survey provides an overview of the European ATMP manufacturing landscape, highlighting critical challenges and growth trends. Strengthening production capacity, harmonization, and cross-institutional collaboration will be essential to enable scalable, compliant, and cost-effective ATMP manufacturing in Europe.

Supplementary video S1A: mCherry-Nalm-6 and BJ-5ta spheroid

Adoptive T-cell therapy is a novel treatment paradigm in which autologous T cells are genetically modified to express a tumor-targeting chimeric antigen receptor (CAR) prior to ex vivo expansion and re-infusion into the patient. Despite remarkable demonstrations of anti-tumor potency in patients with advanced hematological malignancies, long-lasting responses fail to manifest in a substantial fraction of cases. Although several idiosyncratic factors may contribute to the variability in clinical outcomes, there is mounting evidence that the percentage of polyfunctional T cells in the pre-infusion CAR-T cell product strongly correlates with the durability of cancer remission. Unfortunately, standard evaluations of CAR-T cell products currently rely on bulk population measurements or terminal assays, limiting the ability to isolate and study sub-populations with heightened functional properties. Here, we demonstrate a workflow that leverages an optofluidics platform to evaluate both the cytokine secretion profile and activation via CD137 expression of individual CAR-T cells, which can be optionally combined with cytotoxic activity assessment. Cells exhibiting the greatest degree of multimodal functionality can be isolated for further analyses to inform the design of next-generation CAR-T cell therapies.

Profiling CAR T cells presents challenges due to their heterogeneity, complex immune responses, and limited sample availability. Multiplex assays enable functional assessment by allowing the simultaneous detection of a broad range of effector molecules, including cytokines, chemokines, and cytotoxic mediators. Here, we describe a bead-based multiplex assay compatible with flow cytometry for the analysis of effector molecule secretion by human chimeric antigen receptor (CAR)-T cells. CAR T cell supernatants are incubated with a mixture of capture beads comprising multiple fluorescence-coded populations coupled to analyte-specific antibodies. These beads bind soluble targets in the sample, forming bead-analyte complexes that are subsequently detected using labeled detection antibodies. The assay includes a panel of immune mediators such as interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Granzyme B. The use of predefined standards allows quantitative measurement of multiple analytes from a single sample. Data acquisition and analysis can be performed using automated gating approaches provided by instrument-associated or web-based software tools, facilitating data processing. In conclusion, the presented multiplex assay enables multiparametric analysis of CAR T cell functionality and can be applied in contexts such as research, quality control testing, mechanistic studies, and functional characterization.

Supplementary Figure S5. Supplementary data to Figure 4, showing that vitC-CAR19-Ts maintain the activation state without showing signs of exhaustion, including Figure Legend

Supplementary Figure S4. Supplementary data to Figure 3, showing production of effector molecules by (vitC-)CAR19-Ts and (vitC-)CAR19GNLY-KO-Ts upon antigen recognition, including Legend

Despite its great promise, implementation of CAR-T therapy-a personalized, logistically complex, and expensive treatment-remains challenging, hampering patient access across and within countries. Since 2018, six products have been centrally approved in Europe (i.e., the European Economic Area; EU-approved) for 15 hematologic malignancy indications. To better understand patient access to EU-approved commercial CAR-T therapy, we evaluated the current status in all 30 countries where EU-approval is valid plus the UK, addressing economic, clinical, and organizational aspects, and identifying challenges and strategies for improvement. A two-step approach was used, complementing data from marketing authorization holders (4/4 responded) with country-specific insights from clinical experts obtained via an online survey (30/31 responded). In August 2024, 26% of the 31 countries had no CAR-T products commercially available, 74% ≥ 1 product for non-Hodgkin lymphoma and leukemia, and 16% ≥ 1 product for multiple myeloma. One-time payment was the most used reimbursement method. Time to access varied significantly, with medians ranging from 0 (France/Germany) to 53 months (Slovakia). The median number of qualified CAR-T centers per 10 million population per country was 5.0 (IQR: 3.0-6.1). In most countries, patient eligibility assessment was decentralized. Costs and logistical complexity were main factors restricting access in countries with and without commercially available products. Proposed solutions included cost reductions, improving reimbursement processes, and increasing healthcare resources. This study shows that patient access to commercial CAR-T therapy in Europe remains limited. Its insights into this multi-faceted problem can guide policy-making, advocacy work, and research to make this transformative treatment accessible to more patients in need.

Supplementary Figure S2. Supplementary data to Figure 1, showing activation and exhaustion state of (vitC-)UTD-Ts and (vitC-)CAR19-Ts, including Legend

Supplementary Figure 1 illustrates the longitudinal course of myeloma-related parameters throughout the patient’s treatment, as well as the therapeutic response assessed by ^68Ga-Pentixafor PET/CT. Supplementary Figure 2 shows the BCMA-CAR integration sites in the patient’s genome and their distribution Supplementary Figure 3 shows the quality control parameters and the cell type distribution of the whole dataset in the index patient Supplementary Figure 4 shows transcriptomic changes regarding cytotoxicity and IFN-II induced genes as well as activation of (CAR) T cells detected via flow cytometry Supplementary Figure 5 shows inference of CD4+ CAR T cells with other cell types as determined via Cell Phone DB Supplementary Figure 6 illustrates the functional imaging results obtained via the course of treatment Supplementary Figure 7 shows the scRNA-sequencing results from the control group and the parkinson patient in pre-treatment sampels as well as 10 days post treatment. Supplementary Figure 8: Shows the gating strategy of the flow cytometry data.

Supplementary Figure S6. Supplementary data to Figure 7, showing VitC-CAR19-Ts outperforming CAR19-Ts in cytotoxicity against MCTS model, including Figure Legend

Recent advances in chimeric antigen receptor (CAR) T cell therapy have transformed the treatment landscape of multiple myeloma, yet almost all patients ultimately relapse. Chromosomal 1q gains are associated with a higher risk of disease progression and poor prognosis, suggesting that CAR-T targeting of chromosome 1-encoded antigens, such as SLAMF7, may be particularly relevant in advanced disease. However, novel CAR targets raise the risk of on-target, off-tumor toxicities, underscoring the need for controllable CAR-T systems. We systematically assessed pharmacologic and antibody-based strategies to modulate CD19- and SLAMF7-directed CAR-T cells. Tyrosine-kinase inhibitor dasatinib rapidly and reversibly inhibited CAR-T activation, serving as an efficient "on/off" switch with the limitation of also inhibiting unmodified T cells. To surpass this issue, we used antibody-dependent cell cytotoxicity to inhibit CAR-T cells. However, conditioning with fludarabine/cyclophosphamide profoundly depletes NK cells, limiting antibody-dependent CAR-T clearance in patients. Moreover, as NK cells express SLAMF7, they are susceptible to fratricidal cytotoxicity by SLAMF7 CAR-T cells, further reducing this potential off-switch mechanism. To bypass this immune effector cell dependence, we developed a novel strategy using antibody-drug conjugates (ADCs). In this work, we demonstrate that the BCMA-targeting ADC belantamab-mafodotin selectively eliminates BCMA co-expressing CAR-T cells without affecting unmodified T cells. These findings suggest ADCs as a potent, effector cell-independent safety mechanism for CAR-T therapies, potentially enhancing controllability and safety in future clinical applications.

Chimeric antigen receptor (CAR) T cells exhibit high response rates in B-cell malignancies, but most patients eventually relapse. A key mechanism of treatment failure is the loss or downregulation of tumor antigen expression, yet strategies to modulate cell surface levels of CAR T-cell targets remain largely unexplored. Here, we identify B-cell maturation antigen (BCMA), a central CAR T-cell target in multiple myeloma (MM), as a highly shortlived protein that undergoes K48-linked polyubiquitylation at the plasma membrane, leading to its p97-dependent degradation via the ubiquitin-proteasome system (UPS). This previously unprecedented mechanism of plasma membrane protein regulation enables significant enhancement of BCMA expression via proteasome inhibitors (PIs). The clinically approved PI carfilzomib (CFZ) significantly enhances the efficacy of BCMAdirected CAR T cells against both PI-sensitive and-refractory MM cells in vitro and in vivo. Notably, CFZ treatment of 10 patients with BMCA CAR T-cell therapy relapse, under the CFZ after BCMA CAR T-cell (CarCAR) protocol, resulted in increased BCMA expression in all patients. However, clinical responses were observed only in those with residual and/or expanding CAR T cells, suggesting restored CAR T-cell function. These findings provide a rationale for the use of CFZ treatment in relapsed or refractory MM after BCMA CAR T-cell therapy, advocate for future trials combining CFZ with BCMA CAR T cells, and provide a framework for exploring UPS-dependent degradation of other immunotherapy antigens.

Epstein-Barr virus (EBV) infects more than 90% of the population and establishes a lifelong persistence in memory B cells, passing through several latency stages (I-III). In immunocompromised patients, EBV infections and reactivations can lead to severe complications, such as post-transplant lymphoproliferative disorder (PTLD), a malignant B cell lymphoproliferation. The EBV latent membrane protein 2A (LMP2A) induces activation and proliferation of infected B cells and is expressed in latency stages II/III, that are associated with several EBV malignancies. Here, T cell receptor (TCR)-engineered T cells based on a TCR recognizing the clinically relevant HLA-A∗02:01-restricted LMP2A-derived peptide CLGGLLTMV (A∗02_LMP2ACLG) and equipped with a TCR-inducible cassette for IL-18 release (iIL-18_LMP2A_TCR-T cells) aiming to prevent exhaustion and promote remodeling of the immunosuppressive tumor microenvironment (TME) were developed. The iIL-18_LMP2A_TCR-T cells exhibited improved cytotoxicity against HLA-A∗02:01+ EBV-infected B-lymphoblastoid cell lines (EBV+ B-LCLA∗02:01) serving as in vitro PTLD model, when compared to LMP2A_TCR-T cells without iIL-18. The superior functionality of iIL-18_LMP2A_TCR-T cells was further confirmed in multicellular tumor spheroid (MCTS) models, where they mediated sustained control of EBV+ B-LCLA∗02:01 growth, highlighting their potential as an effective therapeutic approach for the immune-mediated eradication of EBV-associated malignancies, including PTLD.

Anaplastic thyroid carcinoma (ATC) is a rare thyroid malignancy with poor prognosis and very limited treatment options. Therefore, the development of novel therapies is urgently needed. Here, we identified the Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1) protein as a specific CAR T cell target for ATC. ROR1 is part of the Wnt signalling pathway, mainly expressed during embryogenesis, but mostly absent in differentiated adult tissue. In ATCs, ROR1 is strongly overexpressed (RNA/protein level, surface expression) and high expression levels are associated with reduced survival. ROR1 CAR Ts specifically target ATC cell lines in 2D and 3D spheroid cultures, reduce the quantity of circulating tumour cells and block tumour metastases in different ATC mouse models. While small tumours were completely eliminated by ROR1 CAR T cells alone, larger tumours required the combination of ROR1 CAR T cells with the multikinase inhibitor lenvatinib. Lenvatinib blocked primary tumour growth, reduced the quantity of immunosuppressive cancer associated fibroblasts (CAF-S1) in the microenvironment and enhanced CAR T cell functionality and activation. Overall, we validated ROR1 as a prime target for CAR T cell therapies in ATC and identified lenvatinib as highly valuable combination partner, which is able to improve CAR T cell functionality.

Recent studies suggest that Chimeric Antigen Receptor (CAR) binding affinity to its ligand affects CAR-T-cell functionality. Affinity engineering towards lower binding strengths might mitigate therapeutic side effects arising from intense CAR-T-cell activation as well as tumor relapse due to antigen-escape or limited persistence of CAR-T cells during sustained activation via high-affinity receptors. Here we characterize a broad range of CARs with varying affinities to the same target epitope and leverage the insights we gain to design a combined high- and low-affinity CAR product. While CAR affinity impacts in vitro functionality minimally, it strongly correlates with tumor control in vivo. Low-affinity binders cause only mild cytokine release syndrome (CRS) in humanized mouse models at the expense of anti-tumour efficiency. In mixtures with low-affinity CARs, high-affinity CARs maintain strong functionality while showing reduced signs of exhaustion and monocyte-induced cytokine production, compared to high-affinity CAR-T cells alone. In long term in vitro and in vivo settings, low-affinity CAR-T cells dominate over time, proving more resilience to chronic antigen exposure. Overall, our findings demonstrate that affinity combination represents a promising strategy to generate more effective CAR-T-cell products with an improved therapeutic index, beyond affinity engineering alone.

Chimeric antigen receptor (CAR) T-cell therapy has shown efficacy in hematologic malignancies but faces challenges in solid tumors and virus-associated malignancies such as posttransplant lymphoproliferative disorder (PTLD). Various strategies, including optimization of receptor design, genetic modifications addressing immunomodulatory mechanisms, and refining the manufacturing process, have been explored to overcome limited in vivo persistence and tumor infiltration, antigen escape, and the immunosuppressive tumor microenvironment. This study investigated the effect of vitamin C (vitC) conditioning on CD19-targeting CAR T cells (vitC-CAR19-T) to improve the efficacy of CAR T-cell therapy. VitC has been shown to influence immune responses through epigenetic regulation and oxidative stress reduction. Enhanced transduction efficiency and proliferative capacity by vitC conditioning resulted in a higher yield of CD4+ and CD8+ CAR19-Ts. VitC-CAR19-Ts exhibited faster and improved cytotoxic response toward CD19+ Nalm-6 cells and Epstein-Barr virus-infected B-lymphoblastoid cell lines, the in vitro model of PTLD. Increased demethylation was observed in TBX21 regions, which was in line with a type 1-like phenotype and higher expression of effector molecules such as granulysin in both CD4+ and CD8+ in vitC-CAR19-Ts, providing insights into the effects of vitC conditioning. Importantly, vitC-CAR19-Ts outperformed CAR19-Ts in long-term antigen stress assays and three-dimensional multicellular spheroid models, indicating a potentially improved in vivo functionality and tumor infiltration capacity. In summary, vitC conditioning represents a promising strategy to enhance CAR T-cell yield, cytotoxic potential, and durability, complementing existing approaches to overcome the limitations of CAR T cells in the treatment of hematologic malignancies and solid tumors.

We report a fatal case of parkinsonism following treatment with ciltacabtagene autoleucel (cilta-cel). To investigate underlying mechanisms, we performed a multipronged longitudinal analysis using single-cell RNA (scRNA)/T-cell receptor (TCR) sequencing, flow cytometry, and cytokine measurements including cerebrospinal fluid (CSF) and peripheral blood (PB) samples, spanning more than 6 months after chimeric antigen receptor (CAR) T-cell therapy. Combined clinical and molecular findings revealed a biphasic immunologic process in the CSF. The early phase was characterized by a selective influx of predominantly CD4+ CAR T cells, accompanied by the evidence of endothelial dysfunction, prior to the clinical manifestation of parkinsonism. A second phase was preceded by a locally restricted inflammatory process in the CSF. Subsequently, an increase in the CSF to serum albumin ratio indicated disruption of the blood-brain barrier, coinciding with a pronounced influx of T cells-primarily CAR T cells but also clonally expanded, cytotoxic CD8+ non-CAR T cells-which was associated with neuronal injury and clinical decline. SIGNIFICANCE:This article examines central nervous system immune dynamics in a patient developing parkinsonism after cilta-cel. A longitudinal real-world dataset of CSF (n = 8) and PB (n = 6) from six matched time points was analyzed using scRNA/TCR sequencing over 6 months, capturing disease onset and progression.

Immunotherapies targeting surface antigens have transformed the treatment landscape of multiple myeloma (MM), with GPRC5D emerging as a promising therapeutic target. Monoallelic loss of GPRC5D is frequently observed in newly diagnosed MM patients, and the incidence of acquired GPRC5D alterations increases following exposure to GPRC5D-directed therapies. However, the functional consequences of both baseline monoallelic and therapy-induced biallelic GPRC5D alterations remain poorly understood. In this study, we modeled monoallelic versus biallelic loss of GPRC5D to investigate their impact on MM cell biology and responsiveness to GPRC5D-targeted immunotherapies. Our results demonstrate that monoallelic GPRC5D loss in OPM-2 cells reduces surface expression of the antigen and confers resistance to GPRC5D-directed therapies. Complete loss of GPRC5D alters the transcriptional state of MM cells and promotes reprogramming of the phosphoproteomic circuitry ultimately resulting in a pro-proliferative chemokine environment. As a result, GPRC5D deficiency increases the basal proliferation rate of MM cells thereby providing a competitive advantage which may further be amplified by selecting these aggressive phenotypes during ongoing treatment with anti-GPRC5D immunotherapies.