Corrigendum: Prediction of severe CRS and determination of biomarkers in B cell-acute lymphoblastic leukemia treated with CAR-T cells.

[This corrects the article DOI: 10.3389/fimmu.2023.1273507.].

Exploration of Thermally Activated Delayed Fluorescence (TADF)-Based Photoredox Catalyst To Establish the Mechanisms of Action for Photodynamic Therapy.

The mechanisms of action (MoA) have been proposed to further reduce the O2 dependence of photodynamic therapy (PDT) significantly. However, the triplet states of traditional photosensitizers are relatively short and also are easily deactivated by the quenching of H2O or O2. This is not conducive for the electron transfer in the photocatalytic process and poses a great obstacle to establish the MoA. Therefore, we selected and synthesized a zirconium(IV) complex (Zr(MesPDPPh)2) reported by Milsmann to address this issue. The specific symmetric and intact geometry endowed Zr(MesPDPPh)2 NPs with long-lived triplet excited state (τ = 350 µs), desired sensitized ability, and improved anti-interfering performance on O2, which was matched with the requirements of photoredox catalyst significantly. The results showed that while PDT (I) and PDT (II) could be achieved simultaneously by leveraging Zr(MesPDPPh)2 NPs, it also could be served as a rare example of thermally activated delayed fluorescence (TADF)-based photoredox catalyst to implement the MoA of PDT. It involved the oxidation of NADH and the establishment of catalytic cycle collaborating by O2 and cytochrome c (cyt c) in normoxia and hypoxia, respectively. As a result, the oxygen-free PDT and tumor-growth inhibition was realized.

Prediction of severe CRS and determination of biomarkers in B cell-acute lymphoblastic leukemia treated with CAR-T cells.

Introduction: CAR-T cell therapy is a novel approach in the treatment of hematological tumors. However, it is associated with life-threatening side effects, such as the severe cytokine release syndrome (sCRS). Therefore, predicting the occurrence and development of sCRS is of great significance for clinical CAR-T therapy. The study of existing clinical data by artificial intelligence may bring useful information. Methods: By analyzing the heat map of clinical factors and comparing them between severe and non-severe CRS, we can identify significant differences among these factors and understand their interrelationships. Ultimately, a decision tree approach was employed to predict the timing of severe CRS in both children and adults, considering variables such as the same day, the day before, and initial values. Results: We measured cytokines and clinical biomarkers in 202 patients who received CAR-T therapy. Peak levels of 25 clinical factors, including IFN-γ, IL6, IL10, ferritin, and D-dimer, were highly associated with severe CRS after CAR T cell infusion. Using the decision tree model, we were able to accurately predict which patients would develop severe CRS consisting of three clinical factors, classified as same-day, day-ahead, and initial value prediction. Changes in serum biomarkers, including C-reactive protein and ferritin, were associated with CRS, but did not alone predict the development of severe CRS. Conclusion: Our research will provide significant information for the timely prevention and treatment of sCRS, during CAR-T immunotherapy for tumors, which is essential to reduce the mortality rate of patients.

Dysbiosis of the Gut Microbiota and Kynurenine (Kyn) Pathway Activity as Potential Biomarkers in Patients with Major Depressive Disorder.

With increasing attention paid to the concept of the microbiota-gut-brain axis, mounting evidence reveals that the gut microbiota is involved in a variety of neurological and psychiatric diseases. However, gut microbiota changes in major depressive disorder (MDD) patients and their association with disease mechanisms remain undefined. Fifty MDD patients and sixty healthy controls were recruited from the Shanghai Healthy Mental Center, China. Fecal samples were collected, and the compositional characteristics of the intestinal flora were determined in MDD patients by MiSeq sequencing. Venous blood was collected for the detection of plasma indoleamine-2,3-dioxygenase (Ido), kynurenine (Kyn) and tryptophan (Trp) levels. Stool samples of bacterial 16S sequencing was carried out. A total of 2,705,809 optimized sequences were obtained, with an average of 54,116 per sample. More unique OTUs were observed at the family, genus and species levels in the control group compared with the MDD cases. Further analysis showed significant changes in the α- and ß-diversities and relative abundance levels of gut microbial entities in MDD patients, as well as elevated amounts of Ido and Kyn indicating Kyn pathway activation, KEGG bacterial 16S function prediction analysis shows a variety of amino acids and metabolic (including Ido, Trp and Kyn) changes in the body of patients with MDD. These may result in increased neurotoxic metabolites and reduced generation of serotonin in the disease process. These changed factors may potentially be utilized as biomarkers for MDD in the future, playing more important roles in the disease course.

A comparison between children and adolescents with autism spectrum disorders and healthy controls in biomedical factors, trace elements, and microbiota biomarkers: a meta-analysis.

Introduction: Autism spectrum disorder (ASD) is a multifaceted developmental condition that commonly appears during early childhood. The etiology of ASD remains multifactorial and not yet fully understood. The identification of biomarkers may provide insights into the underlying mechanisms and pathophysiology of the disorder. The present study aimed to explore the causes of ASD by investigating the key biomedical markers, trace elements, and microbiota factors between children with autism spectrum disorder (ASD) and control subjects. Methods: Medline, PubMed, ProQuest, EMBASE, Cochrane Library, PsycINFO, Web of Science, and EMBSCO databases have been searched for publications from 2012 to 2023 with no language restrictions using the population, intervention, control, and outcome (PICO) approach. Keywords including "autism spectrum disorder," "oxytocin," "GABA," "Serotonin," "CRP," "IL-6," "Fe," "Zn," "Cu," and "gut microbiota" were used for the search. The Joanna Briggs Institute (JBI) critical appraisal checklist was used to assess the article quality, and a random model was used to assess the mean difference and standardized difference between ASD and the control group in all biomedical markers, trace elements, and microbiota factors. Results: From 76,217 records, 43 studies met the inclusion and exclusion criteria and were included in this meta-analysis. The pooled analyses showed that children with ASD had significantly lower levels of oxytocin (mean differences, MD = -45.691, 95% confidence interval, CI: -61.667, -29.717), iron (MD = -3.203, 95% CI: -4.891, -1.514), and zinc (MD = -6.707, 95% CI: -12.691, -0.722), lower relative abundance of Bifidobacterium (MD = -1.321, 95% CI: -2.403, -0.238) and Parabacteroides (MD = -0.081, 95% CI: -0.148, -0.013), higher levels of c-reactive protein, CRP (MD = 0.401, 95% CI: 0.036, 0.772), and GABA (MD = 0.115, 95% CI: 0.045, 0.186), and higher relative abundance of Bacteroides (MD = 1.386, 95% CI: 0.717, 2.055) and Clostridium (MD = 0.281, 95% CI: 0.035, 0.526) when compared with controls. The results of the overall analyses were stable after performing the sensitivity analyses. Additionally, no substantial publication bias was observed among the studies. Interpretation: Children with ASD have significantly higher levels of CRP and GABA, lower levels of oxytocin, iron, and zinc, lower relative abundance of Bifidobacterium and Parabacteroides, and higher relative abundance of Faecalibacterium, Bacteroides, and Clostridium when compared with controls. These results suggest that these indicators may be a potential biomarker panel for the diagnosis or determining therapeutic targets of ASD. Furthermore, large, sample-based, and randomized controlled trials are needed to confirm these results.

Pre-clinical efficacy of CD20-targeted chimeric antigen receptor T cells for non-Hodgkin's lymphoma.

BACKGROUND: A 4-1BB/CD3-ζ-costimulated CAR-T against CD20 (CAR-T20) was subjected to a systemic efficacy evaluation in a cell co-culture model, and NOD-SCID IL-2 receptor gamma null mice (short for NSG mice) were xenografted with human Burkitt's lymphoma Raji cells. METHODS: CAR-T20 cells were incubated with target cells (K562, K562 CD20 or Raji cells) at ratios of 10:1 and 5:1 for 24 h, and the killing rate was estimated by an LDH cytotoxicity assay. To evaluate the effect of CAR-T20 on the survival time of tumor-bearing animals, 30 NSG mice were employed, and Raji-Luc cells (5 × 105 cells per mouse) were administered prior to CAR-T20 administration. The survival time, optical intensity of Raji-Luc cells, clinical symptoms, and body mass of the animals were observed. Another 144 male NSG mice were employed to investigate the proliferation and antitumor effects of CAR-T20. Human cytokine and murine cytokines were detected at 1, 7, 14, 21, 28, 42, 56 and 90 days post-CAR-T administration, while biochemistry index analysis, T-cell and CAR-T-cell detection in peripheral blood, and histopathological examination were performed at 14, 28, 56 and 90 days post-administration. RESULTS: CAR-T20 cells had a specific killing effect on CD20-expressing cells in vitro. At a dose of 1 × 106 per mouse or above, CAR-T20 prolonged the median survival time from 14 days to more than 3 months, inhibited the proliferation of Raji cells in mice, and alleviated the clinical manifestations and weight loss caused by the Raji-Luc cell load. CAR-T20 at a dose of 2 × 106 per mouse or above inhibited the proliferation of Raji cells in mice for up to 111 days post-administration without recurrence. The numbers of T cells and CAR-T cells in the animals administered CAR-T20 increased significantly when Raji cells were markedly proliferated and subsequently decreased when Raji cells were predominantly inhibited. CAR-T20 increased human IFN-γ, murine TNF and murine IL-6 levels and decreased human IL-10 levels in tumor-bearing mice. The incidences of xenografted tumors in organs/tissues were also reduced effectively by CAR-T20. CONCLUSION: The effective dose of CAR-T20 in mice starts from 1 × 106 per mouse, equivalent to a clinical dose of 5 × 106/kg. Together, our data support the clinical translation of CAR-T20 for R/R B-cell NHL patients.

Investigation of CRS-associated cytokines in CAR-T therapy with meta-GNN and pathway crosstalk.

BACKGROUND: Chimeric antigen receptor T-cell (CAR-T) therapy is a new and efficient cellular immunotherapy. The therapy shows significant efficacy, but also has serious side effects, collectively known as cytokine release syndrome (CRS). At present, some CRS-related cytokines and their roles in CAR-T therapy have been confirmed by experimental studies. However, the mechanism of CRS remains to be fully understood. METHODS: Based on big data for human protein interactions and meta-learning graph neural network, we employed known CRS-related cytokines to comprehensively investigate the CRS associated cytokines in CAR-T therapy through protein interactions. Subsequently, the clinical data for 119 patients who received CAR-T therapy were examined to validate our prediction results. Finally, we systematically explored the roles of the predicted cytokines in CRS occurrence by protein interaction network analysis, functional enrichment analysis, and pathway crosstalk analysis. RESULTS: We identified some novel cytokines that would play important roles in biological process of CRS, and investigated the biological mechanism of CRS from the perspective of functional analysis. CONCLUSIONS: 128 cytokines and related molecules had been found to be closely related to CRS in CAR-T therapy, where several important ones such as IL6, IFN-γ, TNF-α, ICAM-1, VCAM-1 and VEGFA were highlighted, which can be the key factors to predict CRS.

CAR T cells equipped with a fully human scFv targeting Trop2 can be used to treat pancreatic cancer.

PURPOSE: Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in treating haematologic malignancies but has not been effective against solid tumours thus far. Trop2 is a tumour-related antigen broadly overexpressed on a variety of tumours and has been reported as a promising target for pancreatic cancers. Our study aimed to determine whether CAR T cells designed with a fully human Trop2-specific single-chain fragment variable (scFv) can be used in the treatment of Trop2-positive pancreatic tumours. METHODS: We designed Trop2-targeted chimeric antigen receptor engineered T cells with a novel human anti-Trop2 scFv (2F11) and then investigated the cytotoxicity, degranulation, and cytokine secretion profiles of the anti-Trop2 CAR T cells when they were exposed to Trop2 + cancer cells in vitro. We also studied the antitumour efficacy and toxicity of Trop2-specific CAR T cells in vivo using a BxPC-3 pancreatic xenograft model. RESULTS: Trop2-targeted CAR T cells designed with 2F11 effectively killed Trop2-positive pancreatic cancer cells and produced high levels of cytotoxic cytokines in vitro. In addition, Trop2-targeted CAR T cells, which persistently circulate in vivo and efficiently infiltrate into tumour tissues, significantly blocked and even eliminated BxPC-3 pancreatic xenograft tumour growth without obvious deleterious effects observed after intravenous injection into NSG mice. Moreover, disease-free survival was efficiently prolonged. CONCLUSION: These results show that Trop2-targeted CAR T cells equipped with a fully human anti-Trop2 scFv could be a potential treatment strategy for pancreatic cancer and could be useful for clinical evaluation.

Consensus on potential biomarkers developed for use in clinical tests for schizophrenia.

Background: Schizophrenia is a serious mental illness affecting approximately 20 million individuals globally. Both genetic and environmental factors contribute to the illness. If left undiagnosed and untreated, schizophrenia results in impaired social function, repeated hospital admissions, reduced quality of life and decreased life expectancy. Clinical diagnosis largely relies on subjective evidence, including self-reported experiences, and reported behavioural abnormalities followed by psychiatric evaluation. In addition, psychoses may occur along with other conditions, and the symptoms are often episodic and transient, posing a significant challenge to the precision of diagnosis. Therefore, objective, specific tests using biomarkers are urgently needed for differential diagnosis of schizophrenia in clinical practice. Aims: We aimed to provide evidence-based and consensus-based recommendations, with a summary of laboratory measurements that could potentially be used as biomarkers for schizophrenia, and to discuss directions for future research. Methods: We searched publications within the last 10 years with the following keywords: 'schizophrenia', 'gene', 'inflammation', 'neurotransmitter', 'protein marker', 'gut microbiota', 'pharmacogenomics' and 'biomarker'. A draft of the consensus was discussed and agreed on by all authors at a round table session. Results: We summarised the characteristics of candidate diagnostic markers for schizophrenia, including genetic, inflammatory, neurotransmitter, peripheral protein, pharmacogenomic and gut microbiota markers. We also proposed a novel laboratory process for diagnosing schizophrenia in clinical practice based on the evidence summarised in this paper. Conclusions: Further efforts are needed to identify schizophrenia-specific genetic and epigenetic markers for precise diagnosis, differential diagnosis and ethnicity-specific markers for the Chinese population. The development of novel laboratory techniques is making it possible to use these biomarkers clinically to diagnose disease.

Feasibility study of a novel preparation strategy for anti-CD7 CAR-T cells with a recombinant anti-CD7 blocking antibody.

Although chimeric antigen receptor (CAR) T cell immunotherapy has shown promising significance in B cell malignancies, success against T cell malignancies remains unsatisfactory because of shared antigenicity between normal and malignant T cells, resulting in fratricide and hindering CAR production for clinical treatment. Here, we report a new strategy of blocking the CD7 antigen on the T cell surface with a recombinant anti-CD7 antibody to obtain a sufficient amount of CD7-targeting CAR-T cells for T cell acute lymphoblastic leukemia (T-ALL) treatment. Feasibility was evaluated systematically, revealing that blocking the CD7 antigen with an antibody effectively blocked CD7-derived fratricide, increased the expansion rate, reduced the proportion of regulatory T (Treg) cells, maintained the stem cell-like characteristics of T cells, and restored the proportion of the CD8+ T cell population. Ultimately, we obtained anti-CD7 CAR-T cells that were specifically and effectively able to kill CD7 antigen-positive target cells, obviating the need for complex T cell modifications. This approach is safer than previous methods and provides a new, simple, and feasible strategy for clinical immunotherapies targeting CD7-positive malignant tumors.

Ruxolitinib reduces severe CRS response by suspending CAR-T cell function instead of damaging CAR-T cells.

The therapeutic effect of CAR-T is often accompanied by sCRS, which is the main obstacle to the promotion of CAR-T therapy. The JAK1/2 inhibitor ruxolitinib has recently been confirmed as clinically effective in maintaining control over sCRS, however, its mechanism remains unclear. In this study, we firstly revealed that ruxolitinib significantly inhibited the proliferation of CAR-T cells without damaging viability, and induced an efficacy-favored differentiation phenotype. Second, ruxolitinib reduced the level of cytokine release not only from CAR-T cells, but also from other cells in the immune system. Third, the cytolytic activity of CAR-T cells was restored once the ruxolitinib was removed; however, the cytokines released from the CAR-T cells maintained an inhibited state to some degree. Finally, ruxolitinib significantly reduced the proliferation rate of CAR-T cells in vivo without affecting the therapeutic efficacy after withdrawal at the appropriate dose. We demonstrated pre-clinically that ruxolitinib interferes with both CAR-T cells and the other immune cells that play an important role in triggering sCRS reactions. This work provides useful and important scientific data for clinicians on the question of whether ruxolitinib has an effect on CAR-T cell function loss causing CAR-T treatment failure when applied in the treatment of sCRS, the answer to which is of great clinical significance.

The differential effects of tumor burdens on predicting the net benefits of ssCART-19 cell treatment on r/r B-ALL patients.

The tumor burden (TB) is significantly related to the severity of cytokine release syndrome (CRS) caused by CAR-T cells, but its correlation with therapeutic efficacy has not been systematically studied. This study focused on the effects of the TB level on both the safety and efficacy of ssCART-19 as a treatment for r/r B-ALL. Taking the 5% tumor burden as the boundary, the study participants were divided into 2 groups, high and low tumor burden groups. Under this grouping strategy, the impacts of differential r/r B-ALL TBs on the clinical therapeutic efficacy (CR rate and long-term survival) and safety profiles after ssCART-19 cell treatment were analysed. 78 patients were reported in this study. The differential B-ALL TBs significantly affected the complete remission (CR) rates of patients treated with ssCART-19, with rates of 93.94% and 75.56% in the low and high TB groups, respectively (P = 0.0358). The effects of TBs on long-term therapeutic efficacy were further studied based on event-free survival (EFS) and overall survival (OS) profiles; both the OS and EFS of the low TB group were better than those of the high TB group, but the differences were not statistically significant. Importantly, the time points of TB measurement did not significantly affect the OS and EFS profiles regardless of whether the TBs were measured before or after fludarabine-cyclophosphamide (FC) preconditional chemotherapy. On the other hand, the severity of CRS was significantly correlated with the TB level (P = 0.0080), and the incidence of sCRS was significantly related to the TB level (the sCRS incidence increased as the TB level increased, P = 0.0224). Unexpectedly, the ssCART-19 cell expansion peaks were not significantly different (P = 0.2951) between the study groups. Patients with a low r/r B-ALL TB yield more net benefits from CAR-T treatment than those with a high TB in terms of safety and CR rate. These findings are critical and valuable for determining the optimal CAR-T cell treatment window for r/r B-ALL patients and will further the development of comprehensive and reasonable CAR-T cell treatment plans for r/r B-ALL patients with differential TBs.Trial registration: ClinicalTrials.gov identifier, NCT03919240.

A Prospective Investigation of Bispecific CD19/22 CAR T Cell Therapy in Patients With Relapsed or Refractory B Cell Non-Hodgkin Lymphoma.

BACKGROUND: The use of T cells expressing chimeric antigen receptor (CAR T) engineered to target CD19 constitutes breakthrough treatment for relapsed or refractory B cell non-Hodgkin lymphoma (R/R B-NHL). Despite improved outcomes, high relapse rate remains a challenge to overcome. Here, we report the clinical results and the pharmacokinetics of bispecific CD19/22 CAR T in patients with R/R B-NHL. METHODS: We performed a prospective, single-arm study of bispecific CD19/22 CAR T cells in R/R B-NHL. We analyzed the safety and efficacy and investigated the kinetic profiles of the CAR T cells. CAR transgene levels were measured using quantitative polymerase chain reaction, and correlation analyses of pharmacodynamic markers and product characteristics, disease conditions, clinical efficacy and adverse events were performed. RESULTS: From August 2017 to September 2020, a total of 32 patients with CD19/22 CAR T administration were analyzed. The overall response rate was 79.3%, and the complete response rate was 34.5%. The progression-free survival (PFS) and overall survival (OS) rates at 12 months were 40.0% and 63.3%, respectively. Among patients who had a CR at 3 months, the PFS and OS rates at 12 months were 66.7% and 100%, respectively. Severe cytokine release syndrome (sCRS) (grade 3 and higher) occurred in nine patients (28.1%). Grade 3 or higher neurologic events occurred in four patients (12.5%). One patient died from irreversible severe CRS-associated acute kidney injury. Long-term CAR T cells persistence correlated with clinical efficacy (133 days vs 22 days, P = 0.004). Patients treated with more than three prior therapies and presenting extranodal organ involvement had lower maximal concentration (Cmax) values than other patients. Responders had higher Cmax and area under the curve values than non-responders. Tumour burden and Cmax were potentially associated with the severity of CRS. CONCLUSIONS: This study demonstrates the safety and potential clinical efficacy of bispecific CD19/22 CAR T cells in patients with R/R B-NHL and highlights the importance of measuring kinetic parameters in PB to predict efficacy and safety in clinical applications of CAR T cell therapy. CLINICAL TRIAL REGISTRATION: https://www.clinicaltrials.gov/ct2/show/NCT03196830, identifier NCT03196830.

Case Report: Reversible Neurotoxicity and a Clinical Response Induced by BCMA-Directed Chimeric Antigen Receptor T Cells Against Multiple Myeloma With Central Nervous System Involvement.

Isolated central nervous system involvement in multiple myeloma (CNS-MM) is rare and carries extremely poor prognosis. Chimeric antigen receptor T cell therapy (CART) targeting B-cell maturation antigen (BCMA) is demonstrated as a promising strategy in MM treatment, but the clinical safety and efficacy of BCMA-CART against isolated CNS-MM remain elusive. Here we report on a 56-year-old male with refractory isolated CNS-MM who received autologous BCMA-CART therapy and developed grade 4 neurological complications. Cerebrospinal fluid (CSF) analyses showed significant expansion of CART cells and a substantially elevated interleukin-6 (IL-6) level. Intravenous methylprednisolone was administered and the symptoms resolved gradually. Unexpectedly, the level of IL-6 in the CSF was maintained for another 3 days even after the relief of the neurological symptoms. A partial response was achieved and sustained for 5.5 months. This is the first report describing a patient with isolated CNS-MM treated using BCMA-CART therapy. The results demonstrated that BCMA-CART cells administered intravenously trafficked into the CSF, eradicated tumor cells, and induced severe but reversible neurological adverse events. This single-patient report suggests that BCMA-CART therapy can be considered as an alternative option for isolated CNS-MM. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03196414.

Preclinical efficacy and safety evaluation of interleukin-6-knockdown CAR-T cells targeting at CD19.

BACKGROUND: ssCART-19 cells with shRNA-IL-6 gene knockdown were subjected to a comprehensive safety evaluation, including efficacy, toxicity and biodistribution studies in NSG (PrkdcscidIL2rgtm1 /Bcgen) mice. METHODS: NSG mice were administered Raji-Luc and then singly dosed with ssCART-19 cells via intravenous infusion. ssCART-19 DNA fragments were quantified in different tissues by qPCR, and the optical intensity of Raji-Luc was determined for evaluate the efficacy of regular CAR-T and ssCART-19 cells. In toxicity study, clinical symptoms observation, body weight measurements, serum biochemical analysis, human cytokine detection, lymphocytes subsets quantification, necropsy and histopathological examination were performed. RESULTS: The ssCART-19 DNA was mainly concentrated in the liver within 3 hours, and was widely distributed in most of the organs/tissues for 4 weeks after administration. Chimeric antigen receptor gene modified T cells (CAR-Ts) were detected in the peripheral blood with a significant increase in number beginning at approximately 3 weeks. ssCART-19 administration resulted in increased of interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin-2 (IL-2), and IL-17A and decreased IL-10 and IL-6 levels. ssCART-19 inhibited the proliferation of Raji-Luc cells in tumor-bearing NSG mice, and reduced the incidence of lymphomas in the liver, kidneys and spleen. It alleviated clinical symptoms caused by tumor cell proliferation in treated animals. CONCLUSIONS: ssCART-19 prolongs the survival time of tumor-bearing mice without obvious risks of immunotoxicity and tumorigenicity. ssCART-19 DNA was found in the brains of treated animals, however no significant central nervous system toxicity was observed. These data were used to support an investigational new drug (IND) application of ssCART-19 for clinical trial in China.

Interleukin-6-knockdown of chimeric antigen receptor-modified T cells significantly reduces IL-6 release from monocytes.

BACKGROUND: T cells expressing a chimeric antigen receptor (CAR) engineered to target CD19 can treat leukemia effectively but also increase the risk of complications such as cytokine release syndrome (CRS) and CAR T cell related encephalopathy (CRES) driven by interleukin-6 (IL-6). Here, we investigated whether IL-6 knockdown in CART-19 cells can reduce IL-6 secretion from monocytes, which may reduce the risk of adverse events. METHODS: Supernatants from cocultures of regular CART-19 cells and B lymphoma cells were added to monocytes in vitro, and the IL-6 levels in monocyte supernatants were measured 24 h later. IL-6 expression was knocked down in regular CART-19 cells by adding a short hairpin RNA (shRNA) (termed ssCART-19) expression cassette specific for IL-6 to the conventional CAR vector. Transduction efficiency and cell proliferation were measured by flow cytometry, and cytotoxicity was measured by evaluating the release of lactate dehydrogenase into the medium. Gene expression was assessed by qRT-PCR and RNA sequencing. A xenograft leukemia mouse model was established by injecting NOD/SCID/γc-/- mice with luciferase-expressing B lymphoma cells, and then the animals were treated with regular CART-19 cells or ssCART-19. Tumor growth was assessed by bioluminescence imaging. RESULTS: Both recombinant IL-6 and CART-19 derived IL-6 significantly triggered IL-6 release by monocytes. IL-6 knockdown in ssCART-19 cells dramatically reduced IL-6 release from monocytes in vitro stduy. In vivo study further demonstrated that the mice bearing Raji cells treated with ssCART-19 cells showed significant lower IL-6 levels in serum than those treated with regular CART-19 cells, but comparable anti-tumor efficacy between the animal groups. CONCLUSION: CAR T-derived IL-6 is one of the most important initiators to amplify release of IL-6 from monocytes that further drive sCRS development. IL-6 knockdown in ssCART-19 cells by shRNA technology provide a promising strategy to improve the safety of CAR T cell therapy.

Characterization of novel dual tandem CD19/BCMA chimeric antigen receptor T cells to potentially treat multiple myeloma.

BACKGROUND: Treatment with chimeric antigen receptor (CAR)-engineered T cells directed against the B-cell maturation antigen (BCMA) promoted transient recovery from multiple myeloma (MM). However, the absence of this antigen on immature plasma cells may limit the efficacy of this modality and facilitate relapse. The purpose of this study is to characterize a novel CAR that includes both a single-chain variable fragment (scFv)-BCMA and an scFv-CD19 in tandem orientation (tan-CAR) in an attempt to target both BCMA and CD19 expression on MM cells. METHOD: The scFv sequences from the anti-CD19 antibody FMC63 and the anti-BCMA antibody C11D5.3 were ligated in tandem with transmembrane and T-cell signaling domains to generate the tan-CAR construct. Specificity and efficacy of activated tan-CAR T cells were analyzed using in vitro proliferation, cytokine release, and cytolysis assays. We also evaluated the in vivo efficacy with a xenograft mouse model that included target tumor cells that expressed CD19 or BCMA and compared the results to those obtained with conventional CAR T cells. RESULTS: The in vitro studies revealed specific activation of tan-CAR T cells by K562 cells that overexpressed CD19 and/or BCMA. Cell proliferation, cytokine release, and cytolytic activity were all comparable to the responses of single scFv CAR T cells. Importantly, in vivo studies of tan-CAR T cells revealed specific inhibition of tumor growth in the mouse xenograft model that included cells expressing both CD19 and BCMA. Systemic administration of tan-CAR T cells resulted in complete tumor remission, in contrast to the reduced efficacies of BCMA-CAR T and CD19-CAR T alone in this setting. CONCLUSION: We report the successful design and execution of novel tan-CAR T cells that promote significant anti-tumor efficacy against both CD19 and BCMA antigen-positive tumor cells in vitro and in vivo. The data from this study reveal a novel strategy that may help to reduce the rate of relapse in the treatment with single scFv-CAR T cells.

Comparation of CART19 and autologous stem-cell transplantation for refractory/relapsed non-Hodgkin's lymphoma.

BACKGROUND: Autologous stem-cell transplantation (ASCT) is the standard treatment for R/R B-NHL, while chimeric antigen receptor T (CAR-T) therapy targeting CD19 emerges as an alternative strategy. Here we report a comparative analysis of the two strategies in a single center. METHODS: We performed a prospective single-arm study of CAR-T therapy in 29 patients with R/R B-NHL and compared the outcomes with contemporaneous 27 patients who received ASCT. NHL was diagnosed by histopathological assessments, and the safety and efficacy were compared. RESULTS: The CAR-T group exhibited better rates of CR (48.0% vs. 20.8%, P=0.046) and one-year OS (74.4% vs. 44.5%, P=0.044) compared with the ASCT group. Subpopulation analysis showed that patients with IPI scores ≥ 3 achieved significantly higher ORR and CR rates in the CAR-T group than in the ASCT group (ORR: 72.0% vs. 10.0%, P=0.002; CR: 38.9% vs 0% P=0.030, respectively). The most common severe adverse events in the CAR-T group were cytokine release syndrome, neurotoxicity and infection compared with cytopenia, gastrointestinal toxicity and infection in the ASCT group. Additionally, the incidence of non-hematologic severe adverse events (SAEs) was markedly lower in the CAR-T group than in the ASCT group (20.7% vs. 48.1% P=0.030). CONCLUSION: CAR-T therapy exhibited superior clinical outcomes in safety and efficacy over ASCT in patients with R/R B-NHL, suggesting CAR-T may be a recommended alternative to ASCT.

[Target-resequencing to identify microRNA-associated SNP and predict the effect of SNP on microRNA function in colorectal cancer patients].

OBJECTIVE: To identify SNPs in the miRNA genes in colorectal cancer (CRC) patients and to investigate their association with CRC. METHODS: DNAs were isolated from 30 CRC tumor tissues and 30 tumor-adjacent tissues, and subjected to target capture using a custom miRNA chip covering 685 miRNA genes from NimbleGen. The captured DNAs were then sequenced using the Illumina's sequencing technology, and the data were analyzed. RESULTS: We identified 64 SNPs in 43 miRNA genes and most of these SNPs are novel SNPs not reported previously. Prediction of functional consequences of the SNPs using TargetScan and miRSNP showed that SNPs of hsa-mir-1273-G/A, hsa-mir-548h-3-C/U, hsa-mir-1290-A/G, and hsa-mir-1273-C/U resulted in reduction of their mature miRNA abundance. SNPs of hsa-mir-376b-C/G, hsa-mir-604-T/C, hsa-mir-1268-T/G and hsa-mir-146a-C/G resulted in changes in their targeted genes. Finally, we focused on the analysis of SNPs in mir-146a and we found that mir-146a rs1052918 C>G was predicted to promote tumorigenesis via the Wnt signaling pathway. CONCLUSIONS: SNPs in the miRNA genes are important for tumorigenesis. The changes by hsa-mir-146a rs1052918 C>G may result in loss of Wnt, constant activation of the Wnt signaling pathway, and uncontrolled cell proliferation and tumor progression.

Next generation sequencing analysis of miRNAs: MiR-127-3p inhibits glioblastoma proliferation and activates TGF-ß signaling by targeting SKI.

Glioblastoma (GBM) proliferation is a multistep process during which the expression levels of many genes that control cell proliferation, cell death, and genetic stability are altered. MicroRNAs (miRNAs) are emerging as important modulators of cellular signaling, including cell proliferation in cancer. In this study, using next generation sequencing analysis of miRNAs, we found that miR-127-3p was downregulated in GBM tissues compared with normal brain tissues; we validated this result by RT-PCR. We further showed that DNA demethylation and histone deacetylase inhibition resulted in downregulation of miR-127-3p. We demonstrated that miR-127-3p overexpression inhibited GBM cell growth by inducing G1-phase arrest both in vitro and in vivo. We showed that miR-127-3p targeted SKI (v-ski sarcoma viral oncogene homolog [avian]), RGMA (RGM domain family, member A), ZWINT (ZW10 interactor, kinetochore protein), SERPINB9 (serpin peptidase inhibitor, clade B [ovalbumin], member 9), and SFRP1 (secreted frizzled-related protein 1). Finally, we found that miR-127-3p suppressed GBM cell growth by inhibiting tumor-promoting SKI and activating the tumor suppression effect of transforming growth factor-ß (TGF-ß) signaling. This study showed, for the first time, that miR-127-3p and its targeted gene SKI, play important roles in GBM and may serve as potential targets for GBM therapy.