Single-cell sequencing reveals VEGFR as a potential target for CAR-T cell therapy in chordoma.

BACKGROUND: Chordomas are rare osseous neoplasms with a dismal prognosis when they recur. Here we identified cell surface proteins that could potentially serve as novel immunotherapeutic targets in patients with chordoma. METHODS: Fourteen chordoma samples from patients attending Xuanwu Hospital Capital Medical University were subjected to single-cell RNA sequencing. Target molecules were identified on chordoma cells and cancer metastasis-related signalling pathways characterised. VEGFR-targeting CAR-T cells and VEGFR CAR-T cells with an additional TGF-ß scFv were synthesised and their in vitro antitumor activities were evaluated, including in a primary chordoma organoid model. RESULTS: Single-cell transcriptome sequencing identified the chordoma-specific antigen VEGFR and TGF-ß as therapeutic targets. VRGFR CAR-T cells and VEGFR/TGF-ß scFv CAR-T cells recognised antigen-positive cells and exhibited significant antitumor effects through CAR-T cell activation and cytokine secretion. Furthermore, VEGFR/TGF-ß scFv CAR-T cells showed enhanced and sustained cytotoxicity of chordoma cell lines in vitro compared with VRGFR CAR-T cells. CONCLUSIONS: This study provides a comprehensive single-cell landscape of human chordoma and highlights its heterogeneity and the role played by TGF-ß in chordoma progression. Our findings substantiate the potential of VEGFR as a target for CAR-T cell therapies in chordoma which, together with modulated TGF-ß signalling, may augment the efficacy of CAR-T cells.

A CH2CH3 hinge region enhances the cytotoxicity of anti-CD5 CAR-T cells targeting T cell acute lymphoblastic leukemia.

Chimeric antigen receptor T cell (CAR-T) therapies show considerable clinical efficacy in patients with B cell malignancies, but their efficacy is limited in patients with T cell acute lymphoblastic leukemia (T-ALL). CD5 is expressed on ∼85 % of malignant T cells, and CD5-targeting CAR-T cells can exhibit potent antitumor activity against T-ALL. However, optimization of CAR costimulatory endo-, hinge, and transmembrane domains could further increase their expansion and persistence, thereby enhancing their efficacy following exposure to tumor cells. Here we designed CD5-specific CARs with different molecular structures to generate CAR-T cells and investigated their anti-tumor efficacy in vitro and in vivo. CD5 CARs with a 4-1BB costimulatory domain (BB.z) or a CD28 costimulatory domain (28.z) exhibited specific cytotoxicity against CD5+ malignant cells in vitro. However, both failed to prolong the survival of T-ALL xenograft mice. Subsequently, we substituted the 28.z CAR hinge region with CH2CH3, which enhanced the ability of CH2CH3-CD5 CAR-T cells to specifically eradicate T-ALL cells in vitro and in vivo. Furthermore, patient-derived CH2CH3-CD5 CAR-T cells were generated which showed a marked killing effect of CD5-positive acute T-ALL cells in vitro. The anti-tumor activity of CD5 CAR-T cells with a CD28 co-stimulation domain and CH2CH3 hinge region was superior to those with BB.z and 28.z domains. These preclinical data provided new insights into the factors dictating efficacy in T-ALL treatment with CAR-T cells and hold promise for clinical translation.

Therapeutic effects against high-grade glioblastoma mediated by engineered induced neural stem cells combined with GD2-specific CAR-NK.

PURPOSE: High-grade glioblastoma is extremely challenging to treat because of its aggressiveness and resistance to conventional chemo- and radio-therapies. On the contrary, genetic and cellular immunotherapeutic strategies based on the stem and immune cells are emerging as promising treatments against glioblastoma (GBM). We aimed to developed a novel combined immunotherapeutic strategy to improve the treatment efficacy using genetically engineered PBMC-derived induced neural stem cells (iNSCs) expressing HSV-TK and second-generation CAR-NK cells against GBM. METHODS: iNSCs cells expressing HSV-TK (iNSCsTK) and GD2-specific CAR-NK92 (GD2NK92) were generated from PBMC-derived iNSCs and NK92 cell lines, respectively. The anti-tumor effect of iNSCsTK and the combinational therapeutics of iNSCsTK and GD2NK92 were evaluated by GBM cell line using in vitro and in vivo experiments. RESULTS: PBMC-derived iNSCsTK possessed tumor-tropism migration ability in vitro and in vivo, which exhibited considerable anti-tumor activity via bystander effect in the presence of ganciclovir (GCV). iNSCsTK/GCV could slow GBM progression and prolong median survival in tumor-bearing mice. However, the anti-tumor effect was limited to single therapy. Therefore, the combinational therapeutic effect of iNSCsTK/GCV and GD2NK92 against GBM was investigated. This approach displayed a more significant anti-tumor effect in vitro and in xenograft tumor mice. CONCLUSIONS: PBMC-derived iNSCsTK showed a significant tumor-tropic migration and an effective anti-tumor activity with GCV in vitro and in vivo. In addition, combined with GD2NK92, iNSCsTK therapeutic efficacy improved dramatically to prolong the tumor-bearing animal model's median survival.

GD2 CAR-T cells in combination with Nivolumab exhibit enhanced antitumor efficacy.

Glioblastoma (GBM) is a common primary brain tumor with poor clinical prognosis. Although CAR-T therapy has been trialed for treatment of GBM, the outcomes are sub-optimal possibly due to exhaustion of T cells and life-threatening neurotoxicity. To address these issues, a combined therapeutic strategy was tested in the current study using GD2 CAR-T together with Nivolumab - an anti-PD-1 monoclonal antibody. An effector-to-target co-culture system was established to evaluate the short-term and long-term cytotoxicity of CAR-T, as well as to investigate the inhibitory activity and T cell exhaustion associated with the PD-1/PD-L1 signaling pathway. Orthotopic NOD/SCID GBM animal models were generated to evaluate the safety and efficacy of the combined therapeutic strategy at various dosages of GD2 CAR-T with Nivolumab. GD2 CAR-T exhibited significant antigen-specific cytotoxicity in a dose-dependent manner in vitro. The persistence of cytotoxicity of GD2 CAR-T could be enhanced by addition of Nivolumab in the co-culture system. Animal studies suggested that GD2 CAR-T effectively infiltrated into tumor tissue and significantly hampered tumor progression. The optimal therapeutic outcome was obtained via using the medium dosage of CAR-T with Nivolumab, which displayed the highest efficacy in extending the survival up to 60 days. Further investigation of toxicity revealed that high-dosage of GD2 CAR-T could induce tumor apoptosis through p53/caspase-3/PARP signaling pathway. This study suggests that GD2 CAR-T in combination with Nivolumab may offer an improved therapeutic strategy for treatment of GBM.

Long-Term Safety and Efficacy of CD19 Humanized Selective CAR-T Therapy in B-ALL Patients Who Have Previously Received Murine-Based CD19 CAR-T Therapy.

Murine-based CD19 CAR-T (CD19m CAR-T) therapy can lead to a relatively high CR rate when administered to B-ALL patients for the first time. However, the DOR is sub-optimal and a subset of patients even show primary resistance to CD19m CAR-T. To address these issues, we employed a humanized selective CD19CAR-T (CD19hs CAR-T) and evaluated the long-term safety and efficacy of treating 8 R/R B-ALL patients who had relapsed or failed to achieve CR following CD19m CAR-T infusion (Clinical trials' number: ChiCTR1800014761 and ChiCTR1800017439). Of the 8 patients, 7 achieved CR on Day 30 after the 1st infusion of CD19hs CAR-T. The median CRS grade was 1 without significant neurotoxicity seen in any of the 8 patients. The median DOR was 11 months, significantly longer than the DOR following CD19mCAR-T infusions. Anti-CAR antibodies were induced in patients who had received prior CD19m CAR-T infusions but not in those following a single or repeated CD19hsCAR-T treatment, which probably had contributed to the sub-optimal DOR and/or failure of effective response in these patients. CD19hs CAR-T, in contrast, induced low immunogenicity compared with CD19m CAR-T, suggesting that a repeat dosing strategy might be feasible and efficacious for patients who have relapsed and/or show primary resistance to CD19m CAR-T therapy. In this clinical study, CD19hs CAR-T showed a significant clinical efficacy with mild side effect among patients with R/R B-ALL who had previously received CD19m CAR-T. Clinical Trial Registration: https://www.chictr.org.cn/showprojen.aspx?proj=25199 (ChiCTR1800014761). https://www.chictr.org.cn/showproj.aspx?proj=29174 (ChiCTR1800017439).

Fibroblast Growth Factor 9 as a Potential Biomarker for Schizophrenia.

Preclinical and clinical studies have suggested that fibroblast growth factor (FGF) system contributed to the onset and development of schizophrenia (SCZ). However, there was no strong clinical evidence to link an individual FGF with SCZ. In this study, we aim to measure blood FGF9 levels in the patients with SCZ with and/or without medication, and test whether FGF9 has a potential to be a biomarker for SCZ. We recruited 130 patients with SCZ and 111 healthy individuals, and the ELISA and qRT-PCR assays were used to measure serum FGF9 levels in the participants. ELISA assay demonstrated that serum FGF9 protein levels were dramatically reduced in first-episode, drug-free patients, but not in chronically medicated patients when compared to healthy control subjects. Further analysis showed that treatment of the first-episode, drug-free SCZ patients with antipsychotics for 8 weeks significantly increased the serum FGF9 levels. In addition, we found that blood FGF9 mRNA levels were significantly lower in first-onset SCZ patients than controls. Under the receiver operating characteristic curve, the optimal cutoff values for FGF9 protein level as an indicator for diagnosis of drug-free SCZ patients was projected to be 166.4 pg/ml, which yielded a sensitivity of 0.955 and specificity of 0.86, and the area under the curve was 0.973 (95% CI, 0.954-0.993). Furthermore, FGF9 had good performance to discriminate between drug-free SCZ patients and chronically medicated patients, the optimal cutoff value for FGF9 concentration was projected to be 165.035 pg/ml with a sensitivity of 0.86 and specificity of 0.919, and the AUC was 0.968 (95% CI, 0.944, 0.991). Taken together, our results for the first time demonstrated the dysregulation of FGF9 in SCZ, and FGF9 has the potential to be served as a biomarker for SCZ.

Edaravone attenuates H2O2 or glutamate-induced toxicity in hippocampal neurons and improves AlCl3/D-galactose induced cognitive impairment in mice.

Edaravone (Eda) is a free radical scavenger used in clinical trials for the treatment of ischemic stroke and amyotrophic lateral sclerosis. However, how Eda exerts its neuroprotective effects remains to be elucidated. We investigated the neuroprotective effects of Eda in cultured hippocampal neurons and in a mouse model of AlCl3/D-galactose-induced cognitive impairment. Eda protected hippocampal neurons by eliminating H2O2 or glutamate-induced toxicity, leading to decreased cell viability and neurite shortening. Consistently, Eda restored impaired levels of BDNF, FGF2 and their associated signaling axes (including TrkB, p-Akt and Bcl-2) to attenuate neuronal death. In a mouse model of chemically-induced cognitive impairment, Eda restored the levels of BDNF, FGF2 and TrkB/Akt signaling axis to attenuate neuronal apoptosis, thereby ameliorating cognitive impairment. Meanwhile, the pro-inflammation was eliminated due to the restoration of pro-inflammatory factors such as TNF-α, IL-6, IL-1ß, and NOS2. In summary, Eda is an effective drug for protecting neurons from neurotoxic injury. BDNF, FGF2, and their regulated pathways may be potential therapeutic targets for neuroprotection.

Edaravone protects rat astrocytes from oxidative or neurotoxic inflammatory insults by restoring Akt/Bcl-2/Caspase-3 signaling axis.

Astrocytes are the major glia cells in the central nervous system (CNS). Increasing evidence indicates that more than to be safe-guard and supporting cells for neurons, astrocytes play a broad spectrum of neuroprotective and pathological functions. Thus, they are compelling models to decipher mechanistic insights of glia cells to CNS insults and for the development of drugs. Edaravone is a free radical scavenger with the capacity to eliminate hydroxyl radicals and lipid peroxides. In this study, we examined the neuroprotective effects of edaravone in rat astrocytes challenged by hydrogen peroxide (H2O2) or bacterial lipopolysaccharides (LPS), respectively. We discovered that edaravone attenuated H2O2-induced oxidative stress by reactivating the Akt signaling axis and antagonistically restoring the expression of apoptosis associated regulators such as Bcl-2 and Caspase-3. Consistently, inhibition of Akt signaling by LY294002 attenuated the anti-oxidative activity of edaravone. In addition, edaravone mitigated LPS-induced morphological changes in astrocytes and alleviated the inflammatory activation and expression of TNF-α, IL-1ß, IL-6 and NOS2. In summary, our data suggested that edavarone effectively protects astrocytes from oxidative stress or infectious insults, which may pave a new avenue for its application in preclinical research and human disease therapeutics.

Case Report: Humanized Selective CD19CAR-T Treatment Induces MRD-Negative Remission in a Pediatric B-ALL Patient With Primary Resistance to Murine-Based CD19CAR-T Therapy.

Background: CD19 chimeric antigen receptor T cell (CD19CAR-T) has shown great potential to treat acute B cell lymphoblastic leukemia (B-ALL) and B cell lymphoma, and most of anti-CD19 scFv are derived from murine antibody sequences. However, about 10-20% of B-ALL patients exhibit primary resistance to murine-based CD19CAR-T (CD19mCAR-T). Herein, we report that a humanized selective CD19CAR-T (CD19hsCAR-T) may offer a solution to this problem. Case Description: A 10-year old boy was diagnosed with high-risk B-ALL in Mar., 2013, and relapsed in Oct., 2018, after he underwent haplo-identical hematopoietic stem cell transplantation (HSCT) in 2017. The patient then received haplo-identical CD19mCAR-T infusions twice following induction chemotherapy with Vincristine, Dexamethasone and Asparaginase (VDL), but no response was observed. We further treated this patient with CD19hsCAR-T following chemotherapy with Vindesine, Idarubicin, Dexamethasone, and Pegylated Asparaginase (VDLD) plus bortezomib. The patient achieved minimal residual disease-negative (MRDneg) complete remission with incomplete hematopoietic recovery (CRi), and remained in CRi for more than 8 months with manageable side effect. The patient, unfortunately, died of unidentified pulmonary infection on Jan. 25 2020. Conclusion: CD19hsCAR-T may have the potential to induce remission in patients who are primarily refractory to CD19mCAR-T.

Treatment with Humanized Selective CD19CAR-T Cells Shows Efficacy in Highly Treated B-ALL Patients Who Have Relapsed after Receiving Murine-Based CD19CAR-T Therapies.

PURPOSE: CD19 chimeric antigen receptor (CAR)-T therapy has shown impactful results in treatment of B-cell malignancies. However, immune recognition of the murine scFv may render subsequent infusion(s) ineffective. Also, nonselective expansion of both CAR-transduced and nontransduced T cells during the production stage affects the yield and purity of final products. Here, we aim to develop a humanized selective (hs) CD19 CAR to solve the above problems.Experimental Design: A CD19 hsCAR was designed, which incorporated a short selective domain between the humanized heavy chain and light chain. The CAR was examined for its property, and then trialed in 5 highly treated B-ALL patients. RESULTS: hsCAR possessed around 6-fold higher affinity to CD19 versus murine CAR (mCAR). Incubation with selective domain-specific mAbs (SmAb) selectively expanded CAR-transduced T cells, and led to a higher proportion of central memory T cells in the final products. SmAb-stimulated CD19 hsCAR-T cells exhibited superior antitumor cytotoxic functions in vitro and in vivo. Autologous (n = 2) and allogeneic donor (n = 3, with hematopoietic stem cell transplantation) hsCAR-T cells were infused into 5 patients who had relapsed after receiving mCAR-T treatments. Two patients received mCAR-T treatments twice previously but the second treatments were ineffective. In contrast, subsequent hsCAR-T treatments proved effective in all 5 patients and achieved complete molecular remission in four, including one with extramedullary disease with central nervous system involvement. CONCLUSIONS: hsCD19 CAR-T treatment shows efficacy in highly treated B-ALL patients who have relapsed after receiving CD19 mCAR-T therapies.

Increased serum FGF2 levels in first-episode, drug-free patients with schizophrenia.

Preclinical and clinical studies suggest that brain-derived neurotrophic factor and nerve growth factor are involved in the pathogenesis of schizophrenia (SCZ). However, the roles of other neurotrophic factors in SCZ remain unclear. The aim of this study was to investigate the blood levels of FGF2 and ADNP in first-episode, drug-free SCZ patients compared with healthy control subjects. 20 SCZ patients, and 20 age and sex matched controls were recruited in this study. Serum FGF2 and ADNP protein levels were measured by ELISA assay, and the results showed that FGF2 levels were significantly increased in patients with SCZ when compared with controls, whereas ADNP protein levels did not significantly associated with SCZ. However, we found that blood ADNP mRNA levels were significantly increased in the patients with SCZ when compared with controls. In addition, subgroup analyses suggested that FGF2 levels were significantly increased in female patients of SCZ, but not in male patients of SCZ. Correlation analyses suggested that age and disease severity (PANSS score) did not have moderating effects on the serum FGF2 levels. Taken together, our results for the first time demonstrated that blood FGF2 was up-regulated in first-episode, drug free-SCZ patients, therefore enhancing the knowledge of neurotrophic factor profile in patients with SCZ.

Postmortem Brain, Cerebrospinal Fluid, and Blood Neurotrophic Factor Levels in Alzheimer's Disease: A Systematic Review and Meta-Analysis.

Accumulating evidence suggest that aberrations of neurotrophic factors are involved in the etiology and pathogenesis of Alzheimer's disease (AD), but clinical data were inconsistent. Therefore, a meta-analysis on neurotrophic factor levels in AD is necessary. We performed a systematic review of blood, CSF, and post-mortem brain neurotrophic factor levels in patients with AD compared with controls and quantitatively summarized the clinical data in blood and CSF with a meta-analytical technique. A systematic search of PubMed and Web of Science identified 98 articles in this study (with samples more than 9000). Random effects meta-analysis demonstrated that peripheral blood BDNF levels were significantly decreased in AD patients compared with controls. However, blood NGF, IGF, and VEGF did not show significant differences between cases and controls. In CSF, random effects meta-analysis showed significantly deceased BDNF and increased NGF levels in patients with AD, whereas IGF and VEGF did not show significant differences between the AD group and control group. In addition, 23 post-mortem studies were included in the systematic review. Although data from post-mortem brains were not always consistent across studies, most studies suggested decreased BDNF and increased (pro)NGF levels in hippocampus and neocortex of patients with AD. These results provide strong clinical evidence that AD is accompanied by an aberrant neurotrophin profile, and future investigations into neurotrophins as biomarkers (especially CSF BDNF and NGF) and therapeutic targets for AD may be warranted.

Neuroprotective effects of a Coeloglossum viride var. Bracteatum extract in vitro and in vivo.

The excessive release and accumulation of glutamate in the brain is known to be associated with excitotoxicity. CE, an extract derived from the plant Coeloglossum viride var. Bracteatum, exerted neuroprotective effects against amyloid toxicity and oxidative stress in cortical neurons. The aims of this study are to examine whether CE also attenuates glutamate neurotoxicity in rat primary cultured cortical neurons and to determine the effect of CE in vivo. According to the results of MTT, LDH release, and TUNEL assays, the CE treatment significantly reduced glutamate-induced neurotoxicity in a dose-dependent manner. Moreover, the protective effects of CE were blocked by an Akt inhibitor, LY294002, suggesting that the PI3K/Akt signalling pathway is involved in the neuroprotective effects of CE. In addition, CE might regulate the PKC-GluA2 axis to prevent neuronal apoptosis. CE also protected against dopaminergic neuronal loss in a mouse model of MPTP-induced PD. Based on our results, CE exerted neuroprotective effects both in vitro and in vivo, thus providing a potential therapeutic target for the treatment or prevention of neurodegeneration.

Dissecting carboxypeptidase E: properties, functions and pathophysiological roles in disease.

Since discovery in 1982, carboxypeptidase E (CPE) has been shown to be involved in the biosynthesis of a wide range of neuropeptides and peptide hormones in endocrine tissues, and in the nervous system. This protein is produced from pro-CPE and exists in soluble and membrane forms. Membrane CPE mediates the targeting of prohormones to the regulated secretory pathway, while soluble CPE acts as an exopeptidase and cleaves C-terminal basic residues from peptide intermediates to generate bioactive peptides. CPE also participates in protein internalization, vesicle transport and regulation of signaling pathways. Therefore, in two types of CPE mutant mice, Cpefat/Cpefat and Cpe knockout, loss of normal CPE leads to a lot of disorders, including diabetes, hyperproinsulinemia, low bone mineral density and deficits in learning and memory. In addition, the potential roles of CPE and ΔN-CPE, an N-terminal truncated form, in tumorigenesis and diagnosis were also addressed. Herein, we focus on dissecting the pathophysiological roles of CPE in the endocrine and nervous systems, and related diseases.

Association of Peripheral Blood Levels of Brain-Derived Neurotrophic Factor With Autism Spectrum Disorder in Children: A Systematic Review and Meta-analysis.

Importance: Accumulating evidence suggests that brain-derived neurotrophic factor (BDNF) may be implicated in the developmental outcomes of children with autism spectrum disorder (ASD). Objective: To use meta-analysis to determine whether children with ASD have altered peripheral blood levels of BDNF. Data Source: A systematic search of PubMed, PsycINFO, and Web of Science was performed for English-language literature through February 7, 2016. The search terms included brain-derived neurotrophic factor or BDNF in combination with autism, without year restriction. Two additional records were retrieved after a review of the reference lists of selected articles. Study Selection: Studies were included if they provided data on peripheral blood levels of BDNF in children with ASD and healthy control children. Studies that included adults or with overlapping samples were excluded. Data Extraction and Synthesis: Data were extracted by 2 independent observers from 19 included studies. Data were pooled using a random-effects model with Comprehensive Meta-analysis software. Main Outcomes and Measures: Blood levels of BDNF in children with ASD compared with healthy controls. Altered levels of BDNF were hypothesized to be related to ASD. Results: This meta-analysis included 19 studies with 2896 unique participants. Random-effects meta-analysis of all 19 studies showed that children with ASD had significantly increased peripheral blood levels of BDNF compared with healthy controls (Hedges g, 0.490; 95% CI, 0.185-0.794; P = .002). Subgroup analyses in 4 studies revealed that neonates diagnosed with ASD later in life had no association with blood levels of BDNF (Hedges g, 0.384; 95% CI, -0.244 to 1.011; P = .23), whereas children in the nonneonate ASD group (15 studies) demonstrated significantly increased BDNF levels compared with healthy controls (Hedges g, 0.524; 95% CI, 0.206 to 0.842; P = .001). Further analysis showed that children in the nonneonate ASD group had increased BDNF levels in serum (10 studies) (Hedges g, 0.564; 95% CI, 0.168 to 0.960; P = .005) but not in plasma (5 studies) (Hedges g, 0.436; 95% CI, -0.176 to 1.048; P = .16). Meta-regression analyses revealed that sample size had a moderating effect on the outcome of the meta-analysis in the nonneonate group. In addition, no publication bias was found in the meta-analysis. Conclusions and Relevance: Children with ASD have increased peripheral blood levels of BDNF, strengthening the clinical evidence of an abnormal neurotrophic factor profile in this population.