Single-cell multiomic dissection of response and resistance to chimeric antigen receptor T cells against BCMA in relapsed multiple myeloma.

Markers that predict response and resistance to chimeric antigen receptor (CAR) T cells in relapsed/refractory multiple myeloma are currently missing. We subjected mononuclear cells isolated from peripheral blood and bone marrow before and after the application of approved B cell maturation antigen-directed CAR T cells to single-cell multiomic analyses to identify markers associated with resistance and early relapse. Differences between responders and nonresponders were identified at the time of leukapheresis. Nonresponders showed an immunosuppressive microenvironment characterized by increased numbers of monocytes expressing the immune checkpoint molecule CD39 and suppressed CD8+ T cell and natural killer cell function. Analysis of CAR T cells showed cytotoxic and exhausted phenotypes in hyperexpanded clones compared to low/intermediate expanded clones. We identified potential immunotherapy targets on CAR T cells, like PD1, to improve their functionality and durability. Our work provides evidence that an immunosuppressive microenvironment causes resistance to CAR T cell therapies in multiple myeloma.

A reliable transcriptomic risk-score applicable to formalin-fixed paraffin-embedded biopsies improves outcome prediction in localized prostate cancer.

BACKGROUND: Clinical manifestation of prostate cancer (PCa) is highly variable. Aggressive tumors require radical treatment while clinically non-significant ones may be suitable for active surveillance. We previously developed the prognostic ProstaTrend RNA signature based on transcriptome-wide microarray and RNA-sequencing (RNA-Seq) analyses, primarily of prostatectomy specimens. An RNA-Seq study of formalin-fixed paraffin-embedded (FFPE) tumor biopsies has now allowed us to use this test as a basis for the development of a novel test that is applicable to FFPE biopsies as a tool for early routine PCa diagnostics. METHODS: All patients of the FFPE biopsy cohort were treated by radical prostatectomy and median follow-up for biochemical recurrence (BCR) was 9 years. Based on the transcriptome data of 176 FFPE biopsies, we filtered ProstaTrend for genes susceptible to FFPE-associated degradation via regression analysis. ProstaTrend was additionally restricted to genes with concordant prognostic effects in the RNA-Seq TCGA prostate adenocarcinoma (PRAD) cohort to ensure robust and broad applicability. The prognostic relevance of the refined Transcriptomic Risk Score (TRS) was analyzed by Kaplan-Meier curves and Cox-regression models in our FFPE-biopsy cohort and 9 other public datasets from PCa patients with BCR as primary endpoint. In addition, we developed a prostate single-cell atlas of 41 PCa patients from 5 publicly available studies to analyze gene expression of ProstaTrend genes in different cell compartments. RESULTS: Validation of the TRS using the original ProstaTrend signature in the cohort of FFPE biopsies revealed a relevant impact of FFPE-associated degradation on gene expression and consequently no significant association with prognosis (Cox-regression, p-value > 0.05) in FFPE tissue. However, the TRS based on the new version of the ProstaTrend-ffpe signature, which included 204 genes (of originally 1396 genes), was significantly associated with BCR in the FFPE biopsy cohort (Cox-regression p-value < 0.001) and retained prognostic relevance when adjusted for Gleason Grade Groups. We confirmed a significant association with BCR in 9 independent cohorts including 1109 patients. Comparison of the prognostic performance of the TRS with 17 other prognostically relevant PCa panels revealed that ProstaTrend-ffpe was among the best-ranked panels. We generated a PCa cell atlas to associate ProstaTrend genes with cell lineages or cell types. Tumor-specific luminal cells have a significantly higher TRS than normal luminal cells in all analyzed datasets. In addition, TRS of epithelial and luminal cells was correlated with increased Gleason score in 3 studies. CONCLUSIONS: We developed a prognostic gene-expression signature for PCa that can be applied to FFPE biopsies and may be suitable to support clinical decision-making.

Cellular dynamics following CAR T cell therapy are associated with response and toxicity in relapsed/refractory myeloma.

B-cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T cells revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM). However, data on cellular (CAR) T cell dynamics and the association with response, resistance or the occurrence of cytokine release syndrome (CRS) are limited. Therefore, we performed a comprehensive flow cytometry analysis of 27 RRMM patients treated with Idecabtagene vicleucel (Ide-cel) to assess the expansion capacity, persistence and effects on bystander cells of BCMA-targeting CAR T cells. Additionally, we addressed side effects, like cytokine release syndrome (CRS) and cytopenia. Our results show that in vivo expansion of CD8+ CAR T cells is correlated to response, however persistence is not essential for durable remission in RRMM patients. In addition, our data provide evidence, that an increased fraction of CD8+ T cells at day of leukapheresis in combination with successful lymphodepletion positively influence the outcome. We show that patients at risk for higher-grade CRS can be identified already prior to lymphodepletion. Our extensive characterization contributes to a better understanding of the dynamics and effects of BCMA-targeting CAR T cells, in order to predict the response of individual patients as well as side effects, which can be counteracted at an early stage or even prevented.

New regulatory thinking is needed for AI-based personalised drug and cell therapies in precision oncology.

Until recently the application of artificial intelligence (AI) in precision oncology was confined to activities in drug development and had limited impact on the personalisation of therapy. Now, a number of approaches have been proposed for the personalisation of drug and cell therapies with AI applied to therapy design, planning and delivery at the patient's bedside. Some drug and cell-based therapies are already tuneable to the individual to optimise efficacy, to reduce toxicity, to adapt the dosing regime, to design combination therapy approaches and, preclinically, even to personalise the receptor design of cell therapies. Developments in AI-based healthcare are accelerating through the adoption of foundation models, and generalist medical AI models have been proposed. The application of these approaches in therapy design is already being explored and realistic short-term advances include the application to the personalised design and delivery of drugs and cell therapies. With this pace of development, the limiting step to adoption will likely be the capacity and appropriateness of regulatory frameworks. This article explores emerging concepts and new ideas for the regulation of AI-enabled personalised cancer therapies in the context of existing and in development governance frameworks.

A time-resolved meta-analysis of consensus gene expression profiles during human T-cell activation.

BACKGROUND: The coordinated transcriptional regulation of activated T-cells is based on a complex dynamic behavior of signaling networks. Given an external stimulus, T-cell gene expression is characterized by impulse and sustained patterns over the course. Here, we analyze the temporal pattern of activation across different T-cell populations to develop consensus gene signatures for T-cell activation. RESULTS: Here, we identify and verify general biomarker signatures robustly evaluating T-cell activation in a time-resolved manner. We identify time-resolved gene expression profiles comprising 521 genes of up to 10 disjunct time points during activation and different polarization conditions. The gene signatures include central transcriptional regulators of T-cell activation, representing successive waves as well as sustained patterns of induction. They cover sustained repressed, intermediate, and late response expression rates across multiple T-cell populations, thus defining consensus biomarker signatures for T-cell activation. In addition, intermediate and late response activation signatures in CAR T-cell infusion products are correlated to immune effector cell-associated neurotoxicity syndrome. CONCLUSION: This study is the first to describe temporally resolved gene expression patterns across T-cell populations. These biomarker signatures are a valuable source, e.g., monitoring transcriptional changes during T-cell activation with a reasonable number of genes, annotating T-cell states in single-cell transcriptome studies, or assessing dysregulated functions of human T-cell immunity.

Characterization and evaluation of gene fusions as a measure of genetic instability and disease prognosis in prostate cancer.

BACKGROUND: Prostate cancer (PCa) is one of the most prevalent cancers worldwide. The clinical manifestations and molecular characteristics of PCa are highly variable. Aggressive types require radical treatment, whereas indolent ones may be suitable for active surveillance or organ-preserving focal therapies. Patient stratification by clinical or pathological risk categories still lacks sufficient precision. Incorporating molecular biomarkers, such as transcriptome-wide expression signatures, improves patient stratification but so far excludes chromosomal rearrangements. In this study, we investigated gene fusions in PCa, characterized potential novel candidates, and explored their role as prognostic markers for PCa progression. METHODS: We analyzed 630 patients in four cohorts with varying traits regarding sequencing protocols, sample conservation, and PCa risk group. The datasets included transcriptome-wide expression and matched clinical follow-up data to detect and characterize gene fusions in PCa. With the fusion calling software Arriba, we computationally predicted gene fusions. Following detection, we annotated the gene fusions using published databases for gene fusions in cancer. To relate the occurrence of gene fusions to Gleason Grading Groups and disease prognosis, we performed survival analyses using the Kaplan-Meier estimator, log-rank test, and Cox regression. RESULTS: Our analyses identified two potential novel gene fusions, MBTTPS2,L0XNC01::SMS and AMACR::AMACR. These fusions were detected in all four studied cohorts, providing compelling evidence for the validity of these fusions and their relevance in PCa. We also found that the number of gene fusions detected in a patient sample was significantly associated with the time to biochemical recurrence in two of the four cohorts (log-rank test, p-value < 0.05 for both cohorts). This was also confirmed after adjusting the prognostic model for Gleason Grading Groups (Cox regression, p-values < 0.05). CONCLUSIONS: Our gene fusion characterization workflow revealed two potential novel fusions specific for PCa. We found evidence that the number of gene fusions was associated with the prognosis of PCa. However, as the quantitative correlations were only moderately strong, further validation and assessment of clinical value is required before potential application.

Four decades of experience of prosthetic valve endocarditis reflect a high variety of diverse pathogens.

Prosthetic valve endocarditis (PVE) remains a serious condition with a high mortality rate. Precise identification of the PVE-associated pathogen/s and their virulence is essential for successful therapy and patient survival. The commonly described PVE-associated pathogens are staphylococci, streptococci, and enterococci, with Staphylococcus aureus being the most frequently diagnosed species. Furthermore, multi-drug resistance pathogens are increasing in prevalence and continue to pose new challenges mandating a personalized approach. Blood cultures in combination with echocardiography are the most common methods to diagnose PVE, often being the only indication, it exists. In many cases, the diagnostic strategy recommended in the clinical guidelines does not identify the precise microbial agent, and frequently, false-negative blood cultures are reported. Despite the fact that blood culture findings are not always a good indicator of the actual PVE agent in the valve tissue, only a minority of re-operated prostheses are subjected to microbiological diagnostic evaluation. In this review, we focus on the diversity and the complete spectrum of PVE-associated bacterial, fungal, and viral pathogens in blood and prosthetic heart valve, their possible virulence potential, and their challenges in making a microbial diagnosis. We are curious to understand if the unacceptable high mortality of PVE is associated with the high number of negative microbial findings in connection with a possible PVE. Herein, we discuss the possibilities and limits of the diagnostic methods conventionally used and make recommendations for enhanced pathogen identification. We also show possible virulence factors of the most common PVE-associated pathogens and their clinical effects. Based on blood culture, molecular biological diagnostics, and specific valve examination, better derivations for the antibiotic therapy as well as possible preventive intervention can be established in the future.

Time to evolve: predicting engineered T cell-associated toxicity with next-generation models.

Despite promising clinical results in a small subset of malignancies, therapies based on engineered chimeric antigen receptor and T-cell receptor T cells are associated with serious adverse events, including cytokine release syndrome and neurotoxicity. These toxicities are sometimes so severe that they significantly hinder the implementation of this therapeutic strategy. For a long time, existing preclinical models failed to predict severe toxicities seen in human clinical trials after engineered T-cell infusion. However, in recent years, there has been a concerted effort to develop models, including humanized mouse models, which can better recapitulate toxicities observed in patients. The Accelerating Development and Improving Access to CAR and TCR-engineered T cell therapy (T2EVOLVE) consortium is a public-private partnership directed at accelerating the preclinical development and increasing access to engineered T-cell therapy for patients with cancer. A key ambition in T2EVOLVE is to design new models and tools with higher predictive value for clinical safety and efficacy, in order to improve and accelerate the selection of lead T-cell products for clinical translation. Herein, we review existing preclinical models that are used to test the safety of engineered T cells. We will also highlight limitations of these models and propose potential measures to improve them.

GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18.

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD2 using the CliniMACS Prodigy® system using a recently described "all-in-one" lentiviral vector combining constitutive anti-GD2 CAR expression and inducible IL-18. Starting with 0.84 x 108 and 0.91 x 108 T cells after enrichment of CD4+ and CD8+ we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 109 and 3.6 x 109 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD2-CAR mediated activation after co-cultivation with GD2-expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD2-expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4+ and CD8+ T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD2-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD2-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.

Taking Lessons from CAR-T Cells and Going Beyond: Tailoring Design and Signaling for CAR-NK Cells in Cancer Therapy.

Cancer immunotherapies utilize the capabilities of the immune system to efficiently target malignant cells. In recent years, chimeric antigen receptor (CAR) equipped T cells showed promising results against B cell lymphomas. Autologous CAR-T cells require patient-specific manufacturing and thus extensive production facilities, resulting in high priced therapies. Along with potentially severe side effects, these are the major drawbacks of CAR-T cells therapies. Natural Killer (NK) cells pose an alternative for CAR equipped immune cells. Since NK cells can be safely transferred from healthy donors to cancer patients, they present a suitable platform for an allogeneic "off-the-shelf" immunotherapy. However, administration of activated NK cells in cancer therapy has until now shown poor anti-cancer responses, especially in solid tumors. Genetic modifications such as CARs promise to enhance recognition of tumor cells, thereby increasing anti-tumor effects and improving clinical efficacy. Although the cell biology of T and NK cells deviates in many aspects, the development of CAR-NK cells frequently follows within the footsteps of CAR-T cells, meaning that T cell technologies are simply adopted to NK cells. In this review, we underline the unique properties of NK cells and their potential in CAR therapies. First, we summarize the characteristics of NK cell biology with a focus on signaling, a fine-tuned interaction of activating and inhibitory receptors. We then discuss why tailored NK cell-specific CAR designs promise superior efficacy compared to designs developed for T cells. We summarize current findings and developments in the CAR-NK landscape: different CAR formats and modifications to optimize signaling, to target a broader pool of antigens or to increase in vivo persistence. Finally, we address challenges beyond NK cell engineering, including expansion and manufacturing, that need to be addressed to pave the way for CAR-NK therapies from the bench to the clinics.

The noncoding RNA LINC00152 conveys contradicting effects in different glioblastoma cells.

Glioblastoma multiforme (GBM) is an extremely aggressive brain tumor, characterized by its high genetic heterogeneity. In search of novel putative therapeutic RNA targets we investigated the role of the oncogenic long noncoding RNA LINC00152 (CYTOR, and STAiR18) in A172 glioblastoma cells. Here, we are the first to describe, that LINC00152 unexpectedly acts in a tumor suppressive manner in this cell line. SiRNA-based knockdown of LINC00152 enhanced malignant tumor behaviors including proliferation, cell cycle entry, migration, and invasion, contradicting previous studies using U87-MG and LN229 glioblastoma cells. Furthermore, LINC00152 knockdown had no influence on survival of A172 glioblastoma cells. In a genome wide transcription analysis of A172 and U87-MG glioblastoma cells, we identified 70 LINC00152 target genes involved in locomotion, cell migration, and motility in A172 cells, whereas in U87-MG cells only 40 target genes were detected. The LINC00152-regulated genes found in A172 differed from those identified in U87-MG glioblastoma cells, none of them being regulated in both cell lines. These findings underline the strong genetic heterogeneity of glioblastoma and point to a potential, yet unknown risk addressing LINC00152 lncRNA as a prospective therapeutic target in GBM.

Distinct Immune Signatures Indicative of Treatment Response and Immune-Related Adverse Events in Melanoma Patients under Immune Checkpoint Inhibitor Therapy.

To identify potential early biomarkers of treatment response and immune-related adverse events (irAE), a pilot immune monitoring study was performed in stage IV melanoma patients by flow cytometric analysis of peripheral blood mononuclear cells (PBMC). Overall, 17 patients were treated with either nivolumab or pembrolizumab alone, or with a combination of nivolumab and ipilimumab every three weeks. Of 15 patients for which complete response assessment was available, treatment responders (n = 10) as compared to non-responders (n = 5) were characterized by enhanced PD-1 expression on CD8+ T cells immediately before treatment (median ± median absolute deviation/MAD 26.7 ± 10.4% vs. 17.2 ± 5.3%). Responders showed a higher T cell responsiveness after T cell receptor ex vivo stimulation as determined by measurement of programmed cell death 1 (PD-1) expression on CD3+ T cells before the second cycle of treatment. The percentage of CD8+ effector memory (CD8+CD45RA-CD45RO+CCR7-) T cells was higher in responders compared to non-responders before and immediately after the first cycle of treatment (median ± MAD 39.2 ± 7.3% vs. 30.5 ± 4.1% and 37.7 ± 4.6 vs. 24.0 ± 6.4). Immune-related adverse events (irAE) were accompanied by a higher percentage of activated CD4+ (CD4+CD38+HLADR+) T cells before the second treatment cycle (median ± MAD 14.9 ± 3.9% vs. 5.3 ± 0.4%). In summary, PBMC immune monitoring of immune-checkpoint inhibition (ICI) treatment in melanoma appears to be a promising approach to identify early markers of treatment response and irAEs.

Detection of Immune Checkpoint Receptors - A Current Challenge in Clinical Flow Cytometry.

Immunological therapy principles are increasingly determining modern medicine. They are used to treat diseases of the immune system, for tumors, but also for infections, neurological diseases, and many others. Most of these therapies base on antibodies, but small molecules, soluble receptors or cells and modified cells are also used. The development of immune checkpoint inhibitors is amazingly fast. T-cell directed antibody therapies against PD-1 or CTLA-4 are already firmly established in the clinic. Further targets are constantly being added and it is becoming increasingly clear that their expression is not only relevant on T cells. Furthermore, we do not yet have any experience with the long-term systemic effects of the treatment. Flow cytometry can be used for diagnosis, monitoring, and detection of side effects. In this review, we focus on checkpoint molecules as target molecules and functional markers of cells of the innate and acquired immune system. However, for most of the interesting and potentially relevant parameters, there are still no test kits suitable for routine use. Here we give an overview of the detection of checkpoint molecules on immune cells in the peripheral blood and show examples of a possible design of antibody panels.

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing.

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these "off-the-shelf" therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

First-in-human intracochlear application of human stromal cell-derived extracellular vesicles.

Extracellular vesicles (EVs) derived from the secretome of human mesenchymal stromal cells (MSC) contain numerous factors that are known to exert anti-inflammatory effects. MSC-EVs may serve as promising cell-based therapeutics for the inner ear to attenuate inflammation-based side effects from cochlear implantation which represents an unmet clinical need. In an individual treatment performed on a 'named patient basis', we intraoperatively applied allogeneic umbilical cord-derived MSC-EVs (UC-MSC-EVs) produced according to good manufacturing practice. A 55-year-old patient suffering from Menière's disease was treated with intracochlear delivery of EVs prior to the insertion of a cochlear implant. This first-in-human use of UC-MSC-EVs demonstrates the feasibility of this novel adjuvant therapeutic approach. The safety and efficacy of intracochlear EV-application to attenuate side effects of cochlea implants have to be determined in controlled clinical trials.

Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL.

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate de novo T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent "induced DCs" (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14+ monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34+ cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.

[CAR T cells as drugs for novel therapies (advanced therapy medicinal products)]. / CAR-T-Zellen als Arzneimittel für neuartige Therapien (Advanced Therapy Medicinal Products).

BACKGROUND: Two commercial chimeric antigen receptor (CAR) T cell products, axicabtagene-ciloleucel (Yescarta®) and tisagenlecleucel (Kymriah®), are registered for the treatment of B cell neoplasia, for which an increased supply of CAR T cell products is required. PROBLEM: The production of patient-specific CAR T cells as advanced therapy medicinal products (ATMPs) poses considerable challenges with respect to logistics, regulation, and manufacturing. METHOD: Review of the CAR T cell manufacturing process and the regulatory network, the current challenges, and future development capabilities of CAR T cells for adoptive immunotherapy. RESULTS: CAR T cells are manufactured under individualized, laborious, good manufacturing practice-conforming processes in decentralized or in specialized centers. Starting from the patient's leukapheresis product, T cells are genetically engineered ex vivo with a CAR, amplified, and after extensive quality control re-applied to the patient. Most CAR T cell products are manufactured in a manual or semi-automated process; fully automated, supervised, and closed systems are increasingly applied to meet the need for a growing number of CAR T cell products. In this setting, research aims at providing allogeneic CAR T cell products or non-T cells such as natural killer cells for broad applications. CONCLUSION: The significance of CAR T cells in adoptive immunotherapy is continuously growing. As individualized cell products, manufacturing requires highly efficient processes under the control of harmonized protocols and regulations so as to ensure the quality of the ATMP in view of increasing demand and to develop new fields in therapy.

Flow cytometric measurement of STAT5 phosphorylation in cytomegalovirus-stimulated T cells.

Cytomegalovirus (CMV)-specific T cells expand with CMV reactivation and are probably prerequisite for control and protection. Given the critical role STAT5A phosphorylation (pSTAT5A) in T cell proliferation, this study presents a simple and sensitive flow cytometric-based pSTAT5A assay to quickly identify CMV-specific T cell proliferation. We determined pSTAT5A in T cells treated with CMV-specific peptide mix (pp65 + IE1 peptides) from 20 healthy adult subjects and three immunodeficient patients with CARMIL-2 mutation. After stimulation, the percentage of pSTAT5A+ T cells in CMV-seropositive (CMV+ ) subjects significantly increased from 3.0% ± 1.9% (unstimulated) to 11.4% ± 5.9% (stimulated) for 24 h. After 7 days of stimulation, the percentage of expanded T cells amounted to 26% ± 17.2%. Conversely, the percentage of pSTAT5A+ T cells and T cell proliferation from CMV-seronegative (CMV- ) subjects hardly changed (from 3.0% ± 1.3% to 3.7% ± 1.8% and from 4.3% ± 2.1% to 5.7% ± 1.7%, respectively). We analyzed the correlation between the percentage of pSTAT5A+ T cells versus (1) CMV-IgG concentrations versus (2) the percentage of expanded T cells and versus (3) the percentage of initial CMV-specific T cells. In immunodeficient patients with CARMIL-2 mutation, CMV-specific pSTAT5A and T cell proliferation were completely deficient. In conclusion, flow cytometric-based pSTAT5A assay represents an appropriate tool to quickly identify CMV-specific T cell proliferation and helps to understand dysfunctions in controlling other pathogens. Flow cytometric-based pSTAT5A assay may be a useful test in clinical practice and merits further validation in large studies.

Extracellular vesicles from human multipotent stromal cells protect against hearing loss after noise trauma in vivo.

The lack of approved anti-inflammatory and neuroprotective therapies in otology has been acknowledged in the last decades and recent approaches are heralding a new era in the field. Extracellular vesicles (EVs) derived from human multipotent (mesenchymal) stromal cells (MSC) can be enriched in vesicular secretome fractions, which have been shown to exert effects (eg, neuroprotection and immunomodulation) of their parental cells. Hence, MSC-derived EVs may serve as novel drug candidates for several inner ear diseases. Here, we provide first evidence of a strong neuroprotective potential of human stromal cell-derived EVs on inner ear physiology. In vitro, MSC-EV preparations exerted immunomodulatory activity on T cells and microglial cells. Moreover, local application of MSC-EVs to the inner ear significantly attenuated hearing loss and protected auditory hair cells from noise-induced trauma in vivo. Thus, EVs derived from the vesicular secretome of human MSC may represent a next-generation biological drug that can exert protective therapeutic effects in a complex and nonregenerating organ like the inner ear.