Chimeric antigen receptor modified T cell (CAR-T) therapy has transformed treatment of a subset of hematological malignancies, driving development of CAR-T products that require robust potency and functional assays. Existing chromium-51, bioluminescence, impedance and flow cytometry assays are either costly, laborious, potentially hazardous and/or lack multiplexing capability. We developed a rapid 3-color image cytometry assay that measures target cell cytotoxicity and CAR-T proliferation, providing an affordable, high-throughput, benchtop alternative to other assays. This novel image cytometry assay and a standard flow cytometry assay demonstrated comparable performance under multiple different conditions when assessing target cell cytotoxicity. Antigen-dependent and -independent CAR-T proliferation and survival were assessed, demonstrating inverse association between antigen-dependent proliferation and the effector:target (E:T) ratio. Compared to a standard flow cytometry assay, preparation and acquisition of 24 samples using this image cytometry assay was markedly faster (164min vs 692 min; 4.2-fold); and labor and materials costs were 25.6% lower than flow cytometry ($265.40 AUD vs $1038.49 AUD). We present a readily available benchtop image cytometry assay that measures CAR-T cytotoxicity and proliferation, and is a more affordable, high-throughput alternative to flow cytometry.
Osteosarcoma (OS) is a rare cancer affecting children and young adults with a good prognosis when localised. Nevertheless, primary metastases or metastatic relapse are associated with high mortality, underlying the need for novel treatment strategies. Immunotherapy, and more recently Chimeric Antigen Receptor (CAR)-based therapy, is being investigated as a new approach for the management of solid tumours, although clinical results in OS have so far been limited. We developed a second-generation CAR (OSCAR-3), derived from the TP-3 hybridoma and targeting ALPL-1, an isoform of alkaline phosphatase selectively expressed in OS. Stable OSCAR-3 expression in T cells demonstrated potent antitumour activity in preclinical models. To enable safer clinical translation, we further engineered OSCAR-3 as an mRNA-based CAR. OSCAR-3 mRNA CAR T cells maintained cytotoxic activity in vitro and in vivo, and, importantly, delayed tumour progression in OS patient-derived xenograft models. These findings support the further development of OSCAR-3 mRNA CAR T cells as a strategic approach that prioritizes safety over persistence for first-in-human studies, while preserving the ability to reduce tumour growth.
PURPOSE:To evaluate the efficacy of a modified protocol involving interleukin-12 (IL-12) and IL-18 in the ex vivo expansion of Vγ9Vδ2 T cells (a major subset of γδ T cells, referred to as GDTs) and to compare the expansion efficiency and functional activity of these cells when peripheral blood (PB) and umbilical cord blood (UCB) were used as cell sources. METHODS:Mononuclear cells obtained from PB and UCB were cultured with zoledronic acid and IL-2 for 15 days. IL-12 was added from days 9-15 and IL-18 was added from days 12-15 to generate advanced GDTs (adGDTs). These adGDTs were compared to conventional GDTs (produced without cytokine supplementation). Expanded cells were then evaluated in terms of phenotype and function, including cytotoxic activity and antigen-presenting capacity. Lastly, associations between baseline lineage composition, including BTN2A1-positive subsets, and GDT yield from UCB were analyzed. RESULTS:In PB-derived cultures, IL-12 and IL-18 supplementation significantly increased GDT cytotoxic activity while maintaining antigen-presenting capacity. With the same protocol, UCB-derived cultures had significantly lower and more variable GDT yields compared with PB-derived cultures (28.13% ± 24.79% versus 54.78% ± 7.73%, P = 0.0007). In the UCB samples, adGDT yield was positively correlated with HLA-DR-positive monocytes (r = 0.7857) and T cells (r = 0.7381) but inversely correlated with the proportions of BTN2A1-positive monocytes (r = -0.6429) and T cells (r = -0.5357). CONCLUSION:IL-12 and IL-18 supplementation generates adGDTs with augmented cytotoxicity without compromising antigen-presenting capacity. PB is a more reliable cell source than is UCB for clinical-scale GDT expansion, and it results in higher and more uniform output under the same protocol.
Background Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by demyelinating lesions leading to cumulative and irreversible neurological damage. In the progressive form of MS (PMS), pathogenic mechanisms are multifaceted, involving both compartmentalized inflammation and neurodegeneration. Owing to their immunomodulatory and trophic properties, mesenchymal stromal cells (MSC) have emerged as a promising therapeutic strategy for this patient population. Objective To review the relevant immunomodulatory and neuroprotective mechanisms of MSC demonstrated in vitro, summarize preclinical and clinical evidence regarding MSC safety and efficacy in experimental autoimmune encephalomyelitis (EAE) murine model and in MS, and discuss key challenges hindering clinical translation. Main results MSC display combined immunomodulatory and neuroprotective properties that are particularly relevant in MS. They induce sustained immune reprogramming of T cells, B cells, and myeloid cells, through paracrine signaling, extracellular vesicle release, metabolic modulation, and efferocytosis of apoptotic cells. These effects are influenced by the local microenvironment, MSC tissue source, manufacturing processes, and route of administration. Beyond immune regulation, MSC promote CNS repair by secreting neurotrophic and angiogenic factors and through mitochondrial transfer. In chronic EAE models, MSC consistently reduce disease severity, mitigate CNS inflammation, and promote neuroprotection, with enhanced efficacy following intrathecal or intraventricular administration. Early-phase clinical trials in PMS have demonstrated a favorable safety profile but inconsistent efficacy, likely reflecting substantial heterogeneity in study design, cell products, dosing regimens, and administration routes. Nevertheless, emerging evidence suggests that repeated intrathecal administration and improved patient stratification may optimize therapeutic outcomes. Conclusion MSC-based therapies represent a potentially valuable avenue for PMS. However, their clinical benefit remains to be firmly established, pending well-designed trials addressing product standardization, optimized delivery approaches, and the use of robust, clinically meaningful endpoints.
Visible particulate matter is a critical quality attribute in injectable drug products; however, current regulatory standards provide limited guidance to address the specific challenges of Cell-Based Advanced Therapies (CT) where conventional inspection and rejection criteria may be incompatible with manufacturing and clinical constraints [1]. Autologous and small-batch therapies often lack manufacturing redundancy, making the rejection of a single unit potentially equivalent to the loss of an opportunity to provide a life-saving treatment. To address this gap, we present a risk-based framework for evaluating and controlling particulate risk in CT, focusing on how particulate sources, process conditions, and control strategies can be characterized and improved early in development while incorporating benefit-risk considerations unique to these therapeutic modalities. Grounded in principles from international quality and regulatory publications, the proposed Visible Particulate Risk Assessment Matrix (VPRAM) guides developers and manufacturers in assessing and managing particulate risk by integrating factors such as particle type and origin, route of administration, material composition, clinical urgency, lot replacement feasibility, and platform-specific experience.
BACKGROUND:Induced pluripotent stem cell (iPSC)-based therapies are rapidly advancing, requiring scalable and standardized cell manufacturing strategies. We established an automated workflow for the expansion of iPSCs and iPSC-derived mesenchymal stromal cells (iMSCs). We evaluated the bioactivities of iPSC- and iMSC-derived extracellular vesicle (EV). METHODS:iPSCs and iMSCs were expanded by the automated culture system through iACE2. EVs were isolated from conditioned media by tangential flow filtration and evaluated for the bioactivities including immunomodulatory phenotypes and anti-senescence effects. RESULTS:Comparable yields of iPSC- and iMSC-derived EVs were isolated from the conditioned medium between automated and manual culture systems. Nanoparticle tracking analysis, transmission electron microscopy, and EV surface marker profiling detected no evident differences in physicochemical properties and bioactivities of EVs between the culture systems. In a radiation-induced fibroblast senescence model, iPSC-derived EVs suppressed a senescent marker CDKN2A/p16INK4A expression, reduced the proportion of senescence-associated β-galactosidase-positive cells, attenuated elevated mitochondrial respiration toward baseline levels, and reduced mitochondrial reactive oxygen species (ROS). We also found that iMSC-derived EVs enhanced mitochondrial respiration in fibroblasts. CONCLUSIONS:Automated culture system through iACE2 supports scalable EV production from iPSCs and iMSCs while preserving EV bioactivity relative to manual procedures. Automated expansion of iPSCs and iMSCs is a practical approach for standardized generation of therapeutically relevant EVs for future translational applications.
Background Solid tumors present unique barriers to treatment with CAR T cells, including poor tumor infiltration into a highly immunosuppressive and metabolically challenging tumor microenvironment (TME). Objectives To enhance both CAR T cell efficacy and the overall immune response against solid tumors, this study explored the therapeutic potential of combining chimeric antigen receptor (CAR) T cells with CD40 stimulation via an agonistic CD40 antibody (αCD40). We hypothesized that CAR T cells could serve as targeted vaccines, promoting antigen release and cooperating with αCD40 to activate and mobilize the endogenous immune cells, thus "heating up" the TME and potentially rendering it more receptive to subsequent therapies. Methods We used a syngeneic mouse model of pancreatic ductal adenocarcinoma (PDAC) and further validated our findings in a triple-negative breast cancer (TNBC) mouse model Results This combined strategy was associated with enhanced anti-tumor activity over CAR T cells alone. This included rapid and sustained tumor necrosis, increased immune cell activation both systemically and within the TME, as well as an overall improvement in survival rates. Comprehensive immune profiling at early timepoints revealed mechanistic insights into the enhanced anti-tumor effects of CAR T cell therapy and αCD40 treatment. Conclusions These findings set the stage for future clinical applications of CAR T cells in combination with CD40 agonists for the treatment of challenging solid tumors.
Immune effector cell-associated neurotoxicity syndrome (ICANS) is a common and life-threatening complication of chimeric antigen receptor (CAR) T-cell therapy, with early detection being critical for timely intervention and improved outcomes. Cytokines such as interleukin-6 (IL-6) are key mediators of the inflammatory cascade underlying ICANS pathogenesis, but prospective clinical evidence establishing whether serial IL-6 elevations precede ICANS onset remains limited. Here we quantify IL-6 levels in a prospective cohort of 40 CAR-T patients (270 serum samples), using a simple in-house microfluidic bead immunoassay. IL-6 levels measured by our assay were significantly associated with next-day ICANS onset. In a generalized estimating equation model adjusted for concurrent CRS grade, each ∼3.4-fold increase in IL-6 levels was linked to a 76% increase in the odds of ICANS onset the following day, independent of other clinical variables. Overall, we show the temporal association of IL-6 with next-day ICANS onset, demonstrate the potential of frequent cytokine measurement to guide CAR-T patient management, and develop a simple experimental method to perform such monitoring.
Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex and costly, and automation holds tremendous promise to improve access to these life-saving treatments. Here, we compared two end-to-end automated CAR T cell manufacturing platforms, the CliniMACS Prodigy® and the Sefia™ system, using identical reagents, stimulation conditions, culture duration, perfusion rate, and donor starting material to assess how platform design affects critical product attributes. With identical CD4 and CD8 magnetic isolation reagents, both systems produced similar T cell yields, with exceptionally high purity and comparable phenotype. During culture, total cell yield differed between platforms, reflecting differences in culture volume; however, expansion rate, population doubling time, transduction efficiency, and CAR T cell yield were not significantly different. The resulting CAR T cells also showed comparable phenotype, exhaustion profiles, and in vitro tumor-killing activity. These similar outcomes may be explained by shared media perfusion rates and convergent metabolic profiles, as cell specific glucose consumption and cell specific lactate production were similar by the end of culture. Collectively, these findings show that distinct automated platforms can generate CAR T cell products with remarkably similar quality and function when process conditions are aligned. More broadly, they highlight the potential of automation and in-line analytics to standardize CAR T cell manufacturing and improve product consistency from cell collection to patient infusion.
Background : TCRαβ/CD19-depleted haploidentical hematopoietic stem cell transplantation (HSCT) is associated with favorable outcomes in pediatric non-malignant disorders when performed in clinically stable patients receiving adequate conditioning. Outcomes in patients transplanted in the setting of active infection remain poorly defined. Objective : To evaluate the impact of pre-transplant infection on outcomes after TCRαβ/CD19-depleted haploidentical HSCT in pediatric non-malignant disorders. Study Design : We retrospectively analyzed 36 pediatric patients with non-malignant disorders who underwent haploidentical HSCT using TCRαβ/CD19-depleted grafts at a single center between 2012 and 2021. Diagnoses included inborn errors of immunity (n=22, including 17 with severe combined immunodeficiency), familial hemophagocytic lymphohistiocytosis (n=9), and bone marrow failure syndromes (n=5). Twenty-two patients (61%) had active infection at transplantation. Results : Engraftment occurred in 26 patients (72%). The cumulative incidence of grade II–III acute graft-versus-host disease was 30%, and chronic graft-versus-host disease occurred in 19%. Fourteen patients died during follow-up. One-year overall survival was 56.1% (95% CI, 41.2–76.6%), with a median follow-up of 20 months. No deaths occurred beyond the first year after transplantation. Infection was the most common cause of death. Survival was significantly higher in patients without infection at transplantation compared with those with active infection (91.7% vs 33%, p=6.5 × 10⁻⁴). Most graft failures occurred in patients who underwent transplantation with reduced-intensity or no conditioning because of severe infection. Conclusion : Pre-transplant infection is strongly associated with inferior survival after TCRαβ/CD19-depleted haploidentical HSCT in pediatric non-malignant disorders. These findings emphasize the importance of infection control, timing of transplantation, and the ability to deliver adequate conditioning.
Mesenchymal stem/stromal cells (MSCs) derived from adipose tissue (AT-MSCs) represent a promising platform for cell-based therapies due to their regenerative and immunomodulatory properties. This study describes the development, validation, and quality control of a clinical-grade, allogeneic MSC-based investigational medicinal product (MSC-IMP) intended for the patients with epidermolysis bullosa (EU CT No. 2024-516614-37-00).A total of eight adipose tissue donations were processed under the Good Manufacturing Practice (GMP) conditions to establish well-characterised intermediate MSC stocks. Three of these stocks were used to manufacture fourteen batches of the final MSC-IMP. All intermediate and final product batches were evaluated for viability, immunophenotype, microbial safety, growth kinetics, colony-forming ability, multilineage differentiation potential, and immunomodulatory properties assessed by mixed lymphocyte reaction (MLR)-based assay. The average yield per 1 ml of lipoaspirate was 0.24 × 10⁶ MSCs in P0. Stock MSCs (P1) demonstrated high viability (mean value 97%), consistent expression of MSC-specific markers (CD73, CD90, CD105 >98%) and negative expression of hematopoietic and endothelial markers. The final MSC-IMP (P2) batches retained excellent viability and maintained functional immunomodulatory properties, exhibiting noimmunostimulatory effect and robust immunosuppressive activity. All final batches met the predefined quality specifications, including microbiological safety, sterility, and the absence of endotoxin. Cryopreserved intermediate stock MSCs remained stable for 24 months with preserved viability, functional and phenotypic properties. A stability study confirmed the stability of fresh, cold-stored MSC-IMP for up to 5 days in HypoThermosol® FRS, with all quality attributes remaining within QC specifications.This study demonstrates a robust and scalable manufacturing process for allogeneic AT-MSCs, yielding a safe, functional, and well-characterised advanced therapy medicinal product suitable for intradermal clinical administration in epidermolysis bullosa or other inflammatory skin conditions.
Background/Aims Neutrophil extracellular traps (NETs) contribute to tissue injury and organ dysfunction in infectious and inflammatory diseases. Although targeting NET formation is an attractive therapeutic strategy, the clinical use of direct NET inhibitors remains uncertain. Mesenchymal stromal cells (MSCs) possess potent immunomodulatory effects, but their functional activity is strongly influenced by culture conditions. We hypothesized that MSCs cultured as three-dimensional (3D) spheroids would suppress endotoxin-induced NET formation more effectively than MSCs expanded in conventional two-dimensional (2D) culture. Methods Bone marrow–derived MSCs (BM-MSCs) and neutrophils were isolated from C57BL/6 mice. Neutrophils were studied under four conditions: unstimulated (NE), lipopolysaccharide-stimulated (NE+LPS), and LPS-stimulated cocultured with either 2D cultured MSCs (NE+LPS+MSC2D) or 3D MSC spheroids (NE+LPS+MSC3D). NET formation was quantified by extracellular DNA release and confirmed by immunofluorescence microscopy. Comparative proteomic analysis was performed to identify candidate mediators underlying the differential immunomodulatory activity of MSC spheroids. Results MSCs cultured as 3D spheroids markedly reduced endotoxin-induced NET formation compared with MSCs grown in 2D culture. This effect was evident by lower extracellular DNA concentrations and reduced NET structures on immunofluorescence microscopy. Proteomic analysis revealed a distinct secretory profile in MSC spheroids enriched for proteins implicated in neutrophil regulation, including SerpinB1, an inhibitor of neutrophil elastase, and developmental endothelial locus-1 (EDIL-3), a mediator of anti-inflammatory signaling. Conclusions A 3D MSC spheroid culture significantly enhances the ability of MSCs to suppress NET formation. This effect is associated with a distinct proteomic signature enriched in neutrophil-modulating proteins. These findings highlight 3D MSC culture as a promising strategy to optimize MSC-based therapies targeting diseases characterized by excessive NET formation.
Macrophages are of interest as candidates for adoptive cell therapy for solid tumors because they can infiltrate tumor tissue, engage target cells, and influence the tumor microenvironment. However, macrophage products derived from primary monocytes remain difficult to standardize, expand, and genetically modify. Pluripotent stem cell-derived myeloid cell lines (PSC-MLs) provide a renewable and engineerable myeloid platform, but their use as direct tumor-targeting effectors has remained incompletely defined.In this study, we generated macrophage-like cells from PSC-MLs carrying a doxycycline-inducible anti-HER2 chimeric antigen receptor (CAR) and evaluated three separable features of the platform: in vitro tumor-cell control, contact-associated and CAR-modified transcriptional responses, and post-administration persistence in vivo. Cells established from 2 human pluripotent stem cell backgrounds retained macrophage-associated phenotypes after differentiation. In co-culture with HER2-expressing tumor cells, the cells showed measurable antitumor activity relative to untreated controls. The DOX-treated condition was compatible with a CAR-associated contribution in some comparisons, but uniform CAR-dependent enhancement was not established across tumor models. Live imaging documented phagocytic events but did not quantify their frequency. Transcriptomic profiling associated tumor-cell contact with inflammatory, trafficking, and uptake-related programs, and the CAR-induced co-culture condition with a relative emphasis on vesicular, lysosomal, and proteostasis-related modules.In a subcutaneous co-implantation assay, in which tumor and effector cells were introduced together, treated groups showed short-term tumor-control signals relative to untreated controls at later observation points. However, an incremental benefit of the CAR-induced (DOX) condition was not clearly resolved. Luciferase-based tracking of ML-MPs showed marked loss by day 3 and no detectable signal by day 7. Repeated dosing was feasible, but its incremental benefit over single dosing could not be established in the small exploratory experiment.
