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.

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.

Supplementary Table 1 summarizes the lines of therapy administered prior to CAR T cell therapy. Supplementary Table 2 presents myeloma parameters and additional laboratory findings obtained immediately before the initiation of lymphodepleting chemotherapy of the index patient. Supplementary Table 3 details the dosing regimens of the drugs used to treat parkinsonism following CAR T cell therapy. Supplementary Table 4 lists the amino acid sequences of the α and β chains of the top expanding T cell clones during parkinsonism. Supplementary Table 5 reports the differentially expressed genes between CSF samples obtained on day 30 and day 43. Supplementary Table 6 reports the differentially expressed genes between CSF samples obtained on day 43 and day 57. Supplementary Table 7 outlines disease characteristics, prior therapies, and CAR T dynamics in the control patient group. Supplementary Table 8 summarizes the baseline disease characteristics of the index patient at primary diagnosis. Supplementary Table 9 provides the markers used in the multiplex assay. Supplementary Table 10 lists the primer sequences used for the CAR integration assay. Supplementary Table 11 compiles the quality-filtering metrics applied to each sample used for single-cell sequencing. Supplementary Table 12 presents all markers identified for each cell cluster in the complete dataset of the index patient. Supplementary Table 13 presents all markers identified for each cell cluster within the T cell subset of the index patient. Supplementary Table 14 summarizes the markers detected in early post-CAR T samples of the index patient and control subjects, as well as in the pre-CAR T samples. Supplementary Table 15 lists the gene signatures used to calculate the IFN-I and IFN-II module scores. Supplementary Table 16 shows the structure of the sequencing data deposited in the European Genome-phenome Archive.

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.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell maturation antigen (BCMA) are transforming treatment for relapsed or refractory multiple myeloma (RRMM). We analyze 61 RRMM patients receiving idecabtagene vicleucel (Ide-cel; n = 34) or ciltacabtagene autoleucel (Cilta-cel; n = 27) and find that Cilta-cel achieves higher complete response (CR) rates (78% vs. 38%) and longer progression-free survival. Using a longitudinal single-cell multi-omics atlas of 135 blood samples, we show that Cilta-cel induces expansion of CD4+ cytotoxic T cells associated with CR and immune-related toxicities, whereas non-CR CD8+ T cells display impaired effector programs. Among non-B cells, plasmacytoid dendritic cells (pDCs) show the highest BCMA expression and BCMA-targeted agents eradicate a blastic plasmacytoid dendritic cell neoplasm line, suggesting a novel therapeutic avenue for this disease. Greater reductions in soluble BCMA correlate with enhanced CAR T expansion and systemic inflammation. These findings reveal cellular mechanisms driving differential efficacy and toxicity of BCMA-directed immunotherapy.

Introduction: BCMA × CD3 bispecific antibodies such as teclistamab induce high response rates and durable remissions in relapsed/refractory multiple myeloma (RRMM). However, due to BCMA expression on normal plasma cells, circulating B cells, and subsets of dendritic cells, these therapies cause profound immunosuppression and hypogammaglobulinemia. While intravenous immunoglobulin replacement can mitigate severe infections to some extent, upper respiratory tract infections remain common. These infections often persist, are prolonged, and significantly impair patients' quality of life. Recent studies in healthy individuals have underscored the importance of nasopharyngeal immunity in protecting against respiratory pathogens such as SARS-CoV-2 (Lindeboom R.G.H. et al. Nature, 2024). Yet, the effect of teclistamab on local mucosal immunity, particularly in the nasopharynx, remains entirely unexplored. Methods: To investigate the impact of teclistamab on nasopharyngeal immunity, we performed deep nasal swabs in 36 RRMM patients treated with teclistamab and 13 RRMM patients receiving daratumumab plus pomalidomide, bortezomib and dexamethasone (control group). In addition, we integrated publicly available single-cell data from healthy individuals from the study of Lindeboom et al. (n = 16). In teclistamab-treated patients, longitudinal sampling was performed at baseline, 1 week, 3 weeks, and 6 months of treatment. Nasopharyngeal cells were dissociated by enzymatic digestion (accutase) and multiple washing steps to generate single-cell suspensions. Cells were subsequently processed for single-cell RNA sequencing using 10x Genomics 5′ chemistry. Results: Single-cell RNA sequencing from nasal swabs was feasible, yielding on average 500 high-quality single cells per sample and the technical success rate of our approach was 90%. Based on canonical marker expression, we identified 17 distinct cell populations, including immune cells (monocytes, CD4⁺ and CD8⁺ T cells, Tregs, NK cells) and epithelial subtypes (ciliated, squamous, ionocytes, basal cells). The cellular composition provided a representative snapshot of the nasopharyngeal microenvironment. Next, we compared the cellular composition of teclistamab- and daratumumab-treated patients to that of healthy individuals. Strikingly, both patient groups completely lacked B cells, and dendritic cells in the nasopharynx, whereas these populations were readily detectable in healthy controls. This suggests a profound disruption of the nasopharyngeal immune barrier in treated myeloma patients, not limited to BCMA-directed therapy. Interestingly, longitudinal sampling in teclistamab-treated patients revealed the emergence of CD4⁺ T cells at later time points expressing TOX2, ENTPD1, BTLA, PDCD1, and CTLA4, a transcriptional signature characteristic of exhausted T cells previously described in autoimmune settings (Saggau C. et al. 2024, Immunity). These cells were barely found in daratumumab-treated patients, suggesting a link between T-cell engaging therapies and the emergence of exhausted T cells in the nasopharyngeal mucosa. We also detected CD8⁺ T cells expressing tissue-residency markers (ZNF683, ITGA1, ITGAE, CD69) that exhibited dynamic transcriptional changes over time and differed from those in daratumumab-treated controls. Additionally, differential gene expression in e.g. WFDC2, SLIT2, SOCS3, RARRES2, was observed in ciliated epithelial cells, supporting the notion that bispecific antibody therapy profoundly impacts on the local mucosa and immune compartment in the nasopharynx. Conclusion: We established a feasible approach to assess local nasopharyngeal immunity in real-world clinical cohorts, revealing teclistamab-associated alterations that may contribute to persistent infections. Ongoing analyses, including an increased sample size as well as samples from patients with viral infections and matched peripheral blood mononuclear cells, will be presented at the meeting.

Circulating tumor plasma cells (CTCs) provide valuable prognostic information and are proposed as a strong factor for risk stratification of multiple myeloma (MM) patients. CD31-expressing plasma cells (PCs) have been described in bone marrow (BM), extramedullary sites, and in peripheral blood (PB), but the prognostic significance of CD31-expressing malignant PCs has remained unclear. Recent reports indicate that Junctional Adhesion Molecule A (JAM-A), expressed on BM malignant PCs is a potential therapeutic target and prognostic factor in MM. Therefore, we investigated CD31 expression on circulating PCs and its correlation with JAM-A expressing CTCs throughout disease stages. Peripheral blood samples from 5 monoclonal gammopathy of undetermined significance (MGUS), 15 smoldering multiple myeloma (SMM), 144 newly diagnosed multiple myeloma (NDMM) patients and 20 healthy controls, BM aspirates from 40 matched MM patients and 7 healthy controls were collected. Using adapted Euroflow protocols and flow cytometry analysis, we analyzed normal and malignant PCs. Data were processed with Infinicyt and Metaflow software and statistically analyzed with Metaflow and GraphPad software. In the group of matched samples, CD31 expression on CTCs was significantly higher in NDMM compared to MGUS (p=0.0061), and significantly higher in SMM compared to MGUS (p=0.0073). The expression of CD31 on normal PCs in healthy controls was significantly lower compared to any myeloma disease stage (p<0.0001 for MGUS, p=0.0001 for SMM and p=0.0001 for NDMM). JAM-A values on CTCs from healthy controls were significantly lower compared to JAM-A levels on CTCs from SMM (p=0.0001) and NDMM (p=0.0009). However, no statistically significant differences in CD31 and JAM-A levels were observed in BM across different groups. We also evaluated JAM-A levels on CTCs at diagnosis in high-risk patients, based on cytogenetic abnormalities defined as t(4;14), del(17p13), t(14;16) or 1qamp/gain (24 with 1q gain/amp, 22 with other HR factors and 98 with standard risk). High JAM-A-expressing CTCs correlated with high-risk (HR) cytogenetic abnormalities. Significant differences were observed between the 1q gain/amp group and patients with other HR factors (p<0.0114), or no cytogenetic abnormalities (p<0.0001). However, JAM-A levels in BM counterparts did not correlate with HR cytogenetic abnormalities. Patients with low JAM-A on CTCs at diagnosis had better prognosis compared to those with high JAM-A-expressing CTCs (n = 67). The median progression-free survival in NDMM with higher JAM-A expression was 19 months, significantly shorter than the 23 months in patients with the lower JAM-A (p=0,0013, Log-rank test). Pearson's correlation analysis revealed that the frequency of JAM-A on CTCs correlated with BM JAM-A levels on malignant PCs in the ND group (Pearson´s r=0.71, p=0.002). In the RRMM setting, CD31+ CTCs strongly correlated with JAM-A+ CTCs (Pearson´s r=0,85, p=0.044) and BM malignant PCs (Pearson´s r=0.52, p=0.084). Additionally, CD31 levels on CTCs correlated with BM CD31 levels on malignant PCs (Pearson´s r=0.62, p=0.03247). CD31 and JAM-A expressing CTCs correlated with clinical parameters such as β2 microglobulin and lactate dehydrogenase in the NDMM group. Collectively, these results underscore the potential of CD31 and JAM-A on CTCs as promising biomarkers for risk stratification in myeloma, in a non-invasive fashion. JAM-A-expressing CTCs are notably associated with cytogenetic risk factors and disease outcomes. The expression of CD31 and JAM-A on CTCs provide insights into CTC biology and conceivable mechanisms of malignant PC dissemination. Beyond prognosis, JAM-A levels on CTCs are linked to cytogenetic risk factors and, thus, they may play a key role in MM patient risk stratification and guiding personalized approaches.

Introduction: The use of ciltacabtagen autoleucel (cilta-cel) in RRMM patients within the CARTITUDE-1 study has exposed previously unknown late onset neurotoxicities, referred to as movement, neurocognitive treatment emergent events (MNT) (Cohen, A et al., 2022). In addition to their late occurrence, MNTs are characterized by their failure to respond to standard therapy such as corticosteroids. Hence, this patient population (5.0 % in CARTITIUDE-1) is at high risk of severe and persistent neurological complications evoking the necessity for further research into the yet poorly understood pathophysiology of MNTs. So far, CAR-T “on-target-off-tumor”-toxicity due to BCMA expression in the basal ganglia has been proposed as one of the driving pathomechanisms of MNTs (Van Oekelen, O et al., 2021). We report the case of a 63-year-old male patient with IgA-kappa myeloma, who received 7 prior lines of treatment. Besides prolonged CRS °I, which was successfully managed by a single dose of tocilizumab, no other adverse events or signs of neurotoxicity occurred immediately post CAR-T infusion. Patient case and methods: 14 days post CAR-T infusion the patient presented with shakiness, subjective slowing of motor skills and concentration disorders. However, it was not until a second hospitalization period 30 days post CAR-T, when neurological examination confirmed a clinical syndrome of parkinsonism including bradykinesia, rigor, tremor and postural instability. Consistent with the clinical presentation, 123I-FP-CIT-SPECT-imaging revealed reduced presynaptic dopamine transporter density in the striatum. On day 54 after CAR-T, the patient developed a massive deterioration of parkinsonism reflected by an increased MDS-UPDRS part III score of 65. Ultimately, the patient was treated with intrathecal chemotherapy, dasatinib orally, cyclophosphamide and corticosteroids. From 149 days post CAR-T a slight improvement of parkinsonism was observed (MDS-UPDRS part III score of 32) following levodopa/benserazide delivery via PEG, and after achieving CAR-T eradication with prior therapies. Multimodal analysis, including flow cytometry, and simultaneous scRNA- and scTCR-seq, was performed on peripheral blood (PB) and cerebrospinal fluid (CSF) longitudinally to the course of illness. Results: CAR-T expansion in PB peaked in the first month post CAR-T (day 17: 7966.7 CAR-T/µl, day 30: 8299.6 CAR-T/µl), while peak infiltration of the CSF occurred at day 57 (367 cells/µl) coinciding with parkinsonism deterioration. Throughout the patient's course, CAR-T were the most abundant cell population in the CSF, with CD4+ CAR-T dominating at the beginning (day 17-30). We performed scRNA-seq and scTCR-seq on 7 longitudinally collected samples (d20-d143 post CAR-T) of the patient's CSF and 6 matched PB samples, collectively representing 74603 cells. CAR-T as well as non-CAR-T cells were present in the CSF with CAR-T proportions decreasing over time. CD8+ as well as CD4+ CAR-T showed marked expression of cytotoxicity associated genes (PRF1, granzymes, GNLY). While CAR-T did not clonally expand, scTCR-seq revealed clonally expanded CD8+ non-CAR-T in the CSF concurring with the deterioration of parkinsonism. Major CD8+ non-CAR-T clones were detectable from day 30 post CAR-T, albeit at low numbers, and were also detectable in the PB. Phenotypically, these clones were marked by the expression of cytotoxicity genes, and tissue residency markers (ZNF683). Cell-cell interaction inference indicated activating signaling of CD4+ CAR-T towards clonally expanded CD8+ non-CAR-T in CSF. Furthermore, we observed an increased interferon response in the CSF that preceded the deterioration of the patient, which was absent in PB. Notably, the interferon response was abrogated after treatment with dasatinib, consistent with reduced activation of CAR-T and non-CAR-T as determined by flow cytometry. Conclusion: Our longitudinal case study shows for the first time that CD4+ CAR-T are most likely the initiators of MNTs and that clonally expanded CD8+ non-CAR-T cells could drive the deterioration of parkinsonism. These findings are consistent with recent reports that CD8+ T cells promote neurodegeneration in other diseases. The coincidence of an interferon response with clinical worsening and the potential abrogation of this response by dasatinib requires further investigation.

Background Emerging evidence has demonstrated rare subacute to late neurological toxicities (NTs) after BCMA CAR T-cell therapy (CAR-T) in multiple myeloma (MM). These adverse events can be categorized as: 1) Parkinsonism (PD), 2) cranial nerve (CN) palsies, and 3) peripheral neuropathies/Guillan Barré syndrome (GBS). These toxicities can cause significant morbidity, but given their relative rarity in clinical trials, there is limited data regarding evaluation and treatment. To our knowledge, this is the largest and only global registry of delayed NTs after BCMA CAR-T. Methods We retrospectively established a registry of NTs via the Immune Therapy Working Committee of the International Myeloma Working Group (IMWG). We gathered data pertaining to demographics, MM characteristics, MM response, NT signs/symptoms, diagnostic workup, and treatment. Result A total of 52 patients with delayed neurotoxicity were included from 14 institutions and 6 countries; 92% received ciltacel, 4% idecel, and 4% investigational CAR-T. Median age at CAR-T infusion was 65 years; 69% were male and 88% were Caucasian. Median bone marrow plasma cell (PC) burden was 15% prior to CAR-T (range 0-95%). Patients received a median of 4 prior lines of therapy (LOT), with 67% progressing on last LOT. Seventeen percent had pre-existing neurological diagnoses (including stroke, epilepsy, migraines). We identified sixteen cases of PD (94% with movement disorder, 69% with cognitive impairment, 75% with personality changes), occurring at a median of 26 days (range: 15-87) after CAR-T. Ninety four percent had CRS (grade (G)1 50%, G2 38%, G3 6%, G4 0%), while 19% had ICANS (G1 13%, G2 6%, G3-G4 0%). Median peak absolute lymphocyte count (ALC) was 9.5 x 109/L, peak C-reactive protein (CRP) 60.9 mg/L, and peak ferritin 1893.5 ug/L within the first 30 days. Concurrently, 3 upper respiratory infections (URIs) and 2 low-level HHV7 in the cerebrospinal fluid (CSF) were detected. A median of 73% of T-cells in the CSF were CAR-T, based on a flow cytometry assay. Ten patients received steroids (median 19 days after symptoms, 70% partial response, 20% no response, 10% unknown), 2 received intrathecal (IT) methotrexate-based therapy (median 32 days after symptoms, 1 with no response, 1 unknown), 5 received intravenous (IV) cyclophosphamide (median 31 days after symptoms, 40% full response, 20% partial response, 40% no response), 4 received high-dose IVIG ≥1g/kg for one or more dose (50% partial response, 50% no response), and 6 received dopamine agonists. There were 37 CN palsies (76% CN7, 15% CN6, 7% CN5, 2% CN3), occurring at a median of 37.5 days (range: 7-482) after CAR-T. Eighty one percent had CRS (G1 68%, G2 14%, G3-G4 0%); 11% had G1 ICANS (G2-G4 0%). Median peak ALC was 4.1 x 109/L, CRP 25.4 mg/L, and ferritin 543 ug/L within the first 30 days. We identified 2 concurrent URIs, 4 CMV viremia (50% with viral load >1500 IU/mL), 1 HHV6 viremia, and 1 HSV in the CSF. Thirty-two (86%) received steroids for a median duration of 12 days, 50% with full response, 3% with no response, and 47% with partial response (of which 43% had later full recovery without further intervention). One received IT methotrexate and another received IV cyclophosphamide without response. Thirteen (41%) received IVIG, of which 38% was high-dose IVIG. Four patients with CN palsies developed peripheral neuropathy/GBS. Eight patients (15%) in this cohort were not heavily pre-treated (≤1 prior LOT); 7 developed CN palsies (6 after ciltacel, 1 after investigational CAR-T) and 1 PD (after idecel). These patients had median <5% PC; none had extramedullary disease. Median ALC was 4.14 x 109/L, CRP 33 mg/L, and ferritin 448.5 ug/L within the first 30 days. Conclusions Delayed NT requires prompt recognition, evaluation for alternative causes, and treatment. In our cohort, the majority of patients were male and Caucasian, suggesting that sex and race may impact the likelihood of developing NT. Individuals with PDs had greater inflammatory response (CRS, ALC, CRP, ferritin) compared to those with CN palsies. CN palsies responded to steroids and/or IVIG, whereas PD had at best partial response to steroids, prompting additional therapy. These toxicities notably did occur in less heavily pre-treated patients, perhaps driven by immune fitness and greater circulating T-cells rather than MM burden. At the time of the annual meeting, data will be updated with additional NTs from less heavily pre-treated patients.

Biomarkers for cytopenias following CAR T-cell treatment in relapsed/refractory (RR) multiple myeloma (MM) are not completely defined. We prospectively analysed 275 sequential peripheral blood (PB) samples from 58 RRMM patients treated with BCMA-targeted CAR T cells, and then divided them into three groups: (i) baseline (before leukapheresis), (ii) ≤day+30, and (iii) >day+30 after CAR T-cell therapy. We evaluated laboratory data and performed flow cytometry to determine the (CAR) T-cell subsets. Baseline hyperferritinaemia was a risk factor for long-lasting grade ≥3 anaemia (r = 0.47, p < 0.001) and thrombocytopenia (r = 0.44, p = 0.002) after CAR T-cell therapy. Low baseline haemoglobin (Hb) and PLT were associated with long-lasting grade ≥3 anaemia (r = -0.56, p < 0.001) and thrombocytopenia (r = -0.44, p = 0.002) respectively. We observed dynamics of CAR-negative T-cell subsets following CAR T-cell infusion. In the late phase after CAR T-cell therapy (>day+30), CD4Tn frequency correlated with anaemia (r = 0.41, p = 0.0014) and lymphocytopenia was related to frequencies of CD8+ T cells (r = 0.72, p < 0.001) and CD8Teff (r = 0.64, p < 0.001). CD4Tcm frequency was correlated with leucocytopenia (r = -0.49, p < 0.001). In summary, preexisting cytopenias and hyperferritinaemia indicated long duration of grade ≥3 post-CAR T-cell cytopenias. Prolonged cytopenia may be related to immune remodelling with a shift in the CAR-negative T-cell subsets following CAR T-cell therapy.

CD19-targeted chimeric antigen receptor-modified T (CAR-T) cells have shown success in clinical studies, with several CD19 CAR-T cell products now having been approved for market use. However, this cell therapy can be associated with side effects such as cytokine release syndrome (CRS). Therefore, pre-clinical test systems are highly desired to permit the evaluation of these unwanted effects before clinical trials begin. In this study, we evaluated cytokine secretion and cell phenotype changes induced by human CD4+ and CD8+ CD19-targeted CAR-T cells in the cytokine release assay (CRA) module of a pre-clinical human in vitro 3D co-culture platform. The in vitro CRA data showed that CD19-targeted CAR-T cells induced a diverse and concentration-dependent cytokine response led by a TH1-profile (IFN gamma, IL-2) and pro-inflammatory cytokines (IL-6, TNF alpha, MCP-1, IL-8, MIP-1b). It was also shown that different cellular components in this 3D co-culture system contributed to the CAR-T cell cytokine response. In particular, whole blood-derived cell populations were necessary to drive the production of T cell cytokines, and endothelial cells were required to generate pro-inflammatory cytokines. CD19-targeted CAR-T cells also triggered cell phenotype changes, including the activation of whole blood-derived CD4+ and CD8+ T-cells and activation/maturation of antigen-presenting cells, during treatment of the in vitro CRA platform. Additionally, the observation of a CD19-targeted CAR-T cell concentration-dependent reduction in the B-cell compartment in this study is aligned with the expected pharmacology and clinical profile of this compound. Overall, this dataset shows the utility of an in vitro CRA model as a pre-clinical platform for evaluating cytokine release potential and analysis of mechanisms of action of CD19-targeted CAR-T cells.

Physiologically relevant human models that recapitulate the challenges of solid tumors and the tumor microenvironment (TME) are highly desired in the chimeric antigen receptor (CAR)-T cell field. We developed a breast cancer-on-chip model with an integrated endothelial barrier that enables the transmigration of perfused immune cells, their infiltration into the tumor, and concomitant monitoring of cytokine release during perfused culture over a period of up to 8 days. Here, we exemplified its use for investigating CAR-T cell efficacy and the ability to control the immune reaction with a pharmacological on/off switch. Additionally, we integrated primary breast cancer organoids to study patient-specific CAR-T cell efficacy. The modular architecture of our tumor-on-chip paves the way for studying the role of other cell types in the TME and thus provides the potential for broad application in bench-to-bedside translation as well as acceleration of the preclinical development of CAR-T cell products.

Although the approval of new drugs has improved the clinical outcome of multiple myeloma (MM), it was widely regarded as incurable over the past decades. However, recent advancements in groundbreaking immunotherapies, such as chimeric antigen receptor T cells (CAR-T), have yielded remarkable results in heavily pretreated relapse/refractory patients, instilling hope for a potential cure. CAR-T are genetically modified cells armed with a novel receptor to specifically recognize and kill tumor cells. Among the potential targets for MM, the B-cell maturation antigen (BCMA) stands out since it is highly and almost exclusively expressed on plasma cells. Here, we review the currently approved BCMA-directed CAR-T products and ongoing clinical trials in MM. Furthermore, we explore innovative approaches to enhance BCMA-directed CAR-T and overcome potential reasons for treatment failure. Additionally, we explore the side effects associated with these novel therapies and shed light on accessibility of CAR-T therapy around the world.

Multiple myeloma (MM) treatment remains challenging due to its relapsed/refractory disease course as well as intra- and inter-patient heterogeneity. Cellular immunotherapies, especially chimeric antigen receptor (CAR)-T cells targeting B cell maturation antigen (BCMA), mark a major breakthrough, achieving long-lasting remissions and instilling hope for a potential cure. While ongoing clinical trials are increasingly driving approved cellular products towards earlier lines of therapy, novel targets as well as advanced approaches employing natural killer (NK) cells or dendritic cell (DC) vaccines are currently under investigation. Treatment resistance, driven by tumor-intrinsic factors such as antigen escape and the intricate dynamics of the tumor microenvironment (TME), along with emerging side effects such as movement and neurocognitive treatment-emergent adverse events (MNTs), are the major limitations of approved cellular therapies. To improve efficacy and overcome resistance, cutting-edge research is exploring strategies to target the microenvironment as well as synergistic combinatorial approaches. Recent advances in CAR-T cell production involve shortened manufacturing protocols and “off-the-shelf” CAR-T cells, aiming at decreasing socioeconomic barriers and thereby increasing patient access to this potential lifesaving therapy. In this review, we provide an extensive overview of the evolving field of cellular therapies for MM, underlining the potential to achieve long-lasting responses.

CD20 is highly expressed in several types of B-cell lymphoma and is an intuitive target for chimeric antigen receptor (CAR) T-cell therapy. However, with conventional approaches, it has been challenging to provide CD20 CAR designs that confer efficacy in preclinical models and in clinical trials. In this issue, Chen and colleagues report several improved CD20 CARs, developed with minimal deviations from conventional design principles, that confer curative anti-lymphoma efficacy in preclinical models. These novel CD20 CARs enrich the pipeline for clinical development and provide an example of rational CAR design that is informed by insights into the structural biology of CAR domains. See related article by Chen et al., p. 150 (3).