Microclimate diversity drives grape quality difference at high-altitude: Observation using PCA analysis and structural equation modeling (SEM).

With the increasing threat of global warming, the cultivation of wine grapes in high-altitude with cool-temperature climates has become a viable option. However, the precise mechanism of environmental factors regulating grape quality remains unclear. Therefore, principal component analysis (PCA) was utilized to evaluate the quality of wine grape (Cabernet Sauvignon) in six high-altitude wine regions (1987, 2076, 2181, 2300, 2430, 2540 m). Structural equation modeling (SEM) was applied for the first time to identify the environmental contribution to grape quality. The wine grape quality existed spatial variation in basic physical attributes (BP), basic chemical compositions (BC), phenolic compounds (PC) and individual phenols. The PCA models (variance > 85 %) well separate wine grapes from the six altitudes into three groups according to scores. The score of grapes at 2300 m was significantly high (3.83), and the grapes of 2540 m showed a significantly low score (1.46). Subsequently, the malic acid, total tannin, total phenol, titratable acid, total anthocyanin, and skin thickness were the main differing indexes. SEM model characterized the relational network of differing indexes and microclimatic factors, which showed that temperature and extreme air temperature had a greater direct effect on differing indexes than light, with great contributions from soil temperature (0.98**), day-night temperature difference (0.825*), and day air temperature (0.789**). Our findings provided a theoretical basis for grape cultivation management in high-altitude regions and demonstrated that the SEM model is a useful tool for exploring the relationship between climate and fruit quality.

Spatial effect of carbon neutrality target on high-quality economic development-Channel analysis based on total factor productivity.

This study utilizes panel data from 30 provinces in mainland China from 2011 to 2020 to investigate the impact of carbon-neutral development on economic high-quality development by constructing an economic high-quality development index and a carbon-neutral development index. Firstly, the study examines the effects of carbon-neutral development on economic high-quality development using baseline regression and spatial Durbin regression. The results indicate that carbon-neutral development has a positive direct effect on economic high-quality growth, but there are negative spatial spillover effects. Secondly, this study employs total factor productivity (TFP) as an intermediate variable in the mediation model regression. The findings demonstrate that carbon-neutral development significantly improves TFP, and the significant improvement in TFP promotes high-quality economic growth. Lastly, the study conducts regional heterogeneity analysis and finds a significant promoting effect of carbon-neutral development on economic high-quality development in the eastern and central regions of China, while it is not significant in the western region. Therefore, it is recommended that China, in the process of achieving carbon-neutral growth, consider the geographical connections between different regions to prevent negative spillover effects. Additionally, regional heterogeneity should be taken into account when formulating relevant policies to promote economic high-quality development.

Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy.

The success of messenger RNA therapeutics largely depends on the availability of delivery systems that enable the safe, effective and stable translation of genetic material into functional proteins. Here we show that extracellular vesicles (EVs) produced via cellular nanoporation from human dermal fibroblasts, and encapsulating mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1) induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. We also show that the intradermal delivery of the mRNA-loaded EVs via a microneedle array led to the prolonged and more uniform synthesis and replacement of collagen in the dermis of the animals. The intradermal delivery of EV-based COL1A1 mRNA may make for an effective protein-replacement therapy for the treatment of photoaged skin.

Impact of environmental regulations on the industrial eco-efficiency in China-based on the strong porter hypothesis and the weak porter hypothesis.

As an important indicator of sustainable development, industrial eco-efficiency (IEE) has aroused growing attention from governments all over the world including China, in recent decades. The Chinese government has introduced numerous environmental regulations; however, the environmental pollution issue does not appear to have been solved. Moreover, although several earlier studies have shown that environmental regulations may promote innovation, there is no consensus on their ultimate effects on IEE. Therefore, this study took a critical look at the connection between environmental regulations and IEE in 36 Chinese sub-sectors from 2009 to 2018. Based on the weak Porter hypothesis (weak PH) and strong Porter hypothesis (strong PH), this paper constructed two panel regression models and conducted group analysis by pollution intensity to check the relationships among environmental regulations, technological innovation, and IEE. It was found that environmental regulations can improve technological innovation and IEE, but these impacts vary across different pollution groups. Specifically, environmental regulations have a U-shaped or inverted U-shaped relationship with technological innovation and IEE. Of the 36 sub-sectors, 26 prove the existence of the Weak PH while 10 verify the Strong PH, indicating that environmental regulations generally advocate technological innovation for most sub-sectors but only promote IEE in a few sub-sectors at present. Finally, differentiated policy implications for environmental regulations and technological innovation are provided for decision-makers.

Polysaccharides from Panax ginseng promote intestinal epithelial cell migration through affecting the Ca2+ related regulators.

Background and aim: Panax ginseng, a key herbal medicine of replenishing Qi and tonifying Spleen, is widely used in the treatment of gastrointestinal diseases in East Asia. In this study, we aim to investigate the potential effects and mechanisms of polysaccharides from P. ginseng (PGP) on intestinal mucosal restitution which is one of the crucial repair modalities during the recovery of mucosal injury controlled by the Ca2+ signaling. Methods: Rat model of intestinal mucosal injury was induced by indomethacin. The fractional cell migration was carried out by immunohistochemistry staining with BrdU. The morphological observations on intestinal mucosal injury were also performed. Intestinal epithelial cell (IEC-6) migration in vitro was conducted by scratch method. Western-blot was adopted to determine the expressions of PLC-γ1, Rac1, TRPC1, RhoA and Cav-1. Immunoprecipitation was used to evaluate the levels of Rac1/PLC-γ1, RhoA/TRPC1 and Cav-1/TRPC1. Results: The results showed that PGP effectively reduced the assessment of intestinal mucosal injury, reversed the inhibition of epithelial cell migration induced by Indomethacin, and increased the level of Ca2+ in intestinal mucosa in vivo. Moreover, PGP dramatically promoted IEC-6 cell migration, the expression of Ca2+ regulators (PLC-γ1, Rac1, TRPC1, Cav-1 and RhoA) as well as protein complexes (Rac1/PLC-γ1, Cav-1/TRPC1 and RhoA/TRPC1) in vitro. Conclusion: PGP increases the Ca2+ content in intestinal mucosa partly through controlling the regulators of Ca2+ mobilization, subsequently promotes intestinal epithelial cell migration, and then prevents intestinal mucosal injury induced by indomethacin.

Effects of the Digital Economy on Carbon Emissions: Evidence from China.

In order to reduce carbon emissions for sustainable development, we analyzed the impact of China's digital economy development on carbon emissions. Based on the panel data of 30 Chinese provinces from 2009 to 2019, we measured the level of development of China's digital economy using the entropy method. The relationship between the digital economy and carbon emissions was analyzed from multiple perspectives with the help of the fixed-effects model, the mediated-effects model and the spatial econometric model. The results indicate that the digital economy plays a significant inhibitory role in carbon emissions. In addition, the digital economy inhibits carbon emissions through the innovation effect and the industrial structure upgrading effect. Moreover, the digital economy exhibits a significant spatial spillover effect in dampening carbon emissions. Finally, there is regional heterogeneity in the direct and spatial spillover effect. The findings provide a basis for the digital economy to contribute to carbon emissions reduction and provide relevant policy references for achieving carbon neutrality and sustainable development.

Activating Connexin43 gap junctions primes adipose tissue for therapeutic intervention.

Adipose tissue is a promising target for treating obesity and metabolic diseases. However, pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization, especially in obese subjects. We have previously shown that during cold exposure, connexin43 (Cx43) gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells. We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue. Using an adipose tissue-specific Cx43 overexpression mouse model, we demonstrate effectiveness in connecting adipocytes to augment metabolic efficacy of the ß 3-adrenergic receptor agonist Mirabegron and FGF21. Additionally, combing those molecules with the Cx43 gap junction channel activator danegaptide shows a similar enhanced efficacy. In light of these findings, we propose a model in which connecting adipocytes via Cx43 gap junction channels primes adipose tissue to pharmacological agents designed to engage it. Thus, Cx43 gap junction activators hold great potential for combination with additional agents targeting adipose tissue.

Upregulated Expression of CYBRD1 Predicts Poor Prognosis of Patients with Ovarian Cancer.

Cytochrome b reductase 1 (CYBRD1) promotes the development of ovarian serous cystadenocarcinoma (OV). We assessed the function of CYBRD1 in OV underlying The Cancer Genome Atlas (TCGA) database. The correlation between clinicopathological characteristics and CYBRD1 expression was estimated. The Cox proportional hazards regression model and the Kaplan-Meier method were applied to identify clinical features related to overall survival and disease-specific survival. Gene set enrichment analysis (GSEA) was applied to identify the relationship between CYBRD1 expression and immune infiltration. CYBRD1 expression in OV was significantly associated with poor outcomes of primary therapy and FIGO stage. Patients with high levels of CYBRD1 expression were prone to the development of a poorly differentiated tumor and experience of an unfavorable outcome. CYBRD1 expression had significant association with shorter OS and acts as an independent predictor of poor outcome. Moreover, enhanced CYBRD1 expression was positively associated with Tem, NK cells, and mast cells but negatively associated with CD56 bright NK cells and Th2 cells. CYBRD1 expression may serve as a diagnostic and prognostic indicator of OV patients. The mechanisms of poor prognosis of CYBRD1-mediated OV may include increased iron uptake, regulation of immune microenvironment, ferroptosis related pathway, and ERK signaling pathway, among which ferroptosis and ERK signaling pathway may be important pathways of CYBRD1-mediated OV. Furthermore, we verified that CYBRD1 was upregulated in OV and significant correlated with lymph nodes metastasis, advanced stage, poor-differentiated tumor, and poor clinical prognosis in East Hospital cohort. The results of this study may provide guidance for the development of optimal treatment strategies for OV.

PERK in POMC neurons connects celastrol with metabolism.

ER stress and activation of the unfolded protein response in the periphery as well as the central nervous system have been linked to various metabolic abnormalities. Chemically lowering protein kinase R-like ER kinase (PERK) activity within the hypothalamus leads to decreased food intake and body weight. However, the cell populations required in this response remain undefined. In the current study, we investigated the effects of proopiomelanocortin-specific (POMC-specific) PERK deficiency on energy balance and glucose metabolism. Male mice deficient for PERK in POMC neurons exhibited improvements in energy balance on a high-fat diet, showing decreased food intake and body weight, independent of changes in glucose and insulin tolerances. The plant-based inhibitor of PERK, celastrol, increases leptin sensitivity, resulting in decreased food intake and body weight in a murine model of diet-induced obesity (DIO). Our data extend these observations by demonstrating that celastrol-induced improvements in leptin sensitivity and energy balance were attenuated in mice with PERK deficiency in POMC neurons. Altogether, these data suggest that POMC-specific PERK deficiency in male mice confers protection against DIO, possibly providing a new therapeutic target for the treatment of diabetes and metabolic syndrome.

IL-15 mediated expansion of rare durable memory T cells following adoptive cellular therapy.

BACKGROUND: Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1-specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression. METHOD: We performed a phase I clinical trial evaluating the safety of NY-ESO-1-specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15. RESULTS: Four patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1-specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor-based products at other centers. CONCLUSIONS: ETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1-specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

Impact of financial development and technological innovation on the volatility of green growth-evidence from China.

China's green growth has shown a trend of fluctuation year by year. Simultaneously, Chinese local governments have pursued simple economic growth driven by the interests of "political competition" for a long time, while the supervision of the ecological environment has been loosened and tightened. In this environment, financial development and technological innovation may easily become the accelerator of this phenomenon, thus exacerbating the fluctuation of green growth. To deeply excavate the key factors to achieve stable and sustained growth of green economy, based on the annual panel data of 30 provinces in China from 2011 to 2018, this paper studies the impact of financial development and technological innovation on the volatility of green growth using dynamic system GMM method. The findings of this paper are shown as follows: First, the expansion of financial institutions' scale will significantly enhance the volatility of green growth. Second, the increase in the scale of the stock market will also significantly cause green growth fluctuations. Third, the interaction between financial development and technological innovation can significantly weaken the volatility of green growth. Fourth, financial development measured by stock market indicators is more efficient than financial development measured by financial institutions indicators to curb the volatility of green growth. Fifth, the fluctuation of green growth in the previous period will reduce the volatility of green growth in the current period. This study provides new evidence for exploring the power source to promote the stability and sustainable growth of the green economy in the special stage of financial and technological integration. Controlling the development scale of financial institutions and removing their state preferences, expanding the development of capital markets, and deepening the integration of financial development and technological innovation are conducive to achieve stable green growth.

Histiocyte predominant myocarditis resulting from the addition of interferon gamma to cyclophosphamide-based lymphodepletion for adoptive cellular therapy.

BACKGROUND: Adoptive cellular therapy (ACT) is a promising treatment for synovial sarcoma (SS) with reported response rates of over 50%. However, more work is needed to obtain deeper and more durable responses. SS has a 'cold' tumor immune microenvironment with low levels of major histocompatibility complex (MHC) expression and few T-cell infiltrates, which could represent a barrier toward successful treatment with ACT. We previously demonstrated that both MHC expression and T-cell infiltration can be increased using systemic interferon gamma (IFN-γ), which could improve the efficacy of ACT for SS. CASE PRESENTATION: We launched a phase I trial incorporating four weekly doses of IFN-γ in an ACT regimen of high-dose cyclophosphamide (HD Cy), NY-ESO-1-specific T cells, and postinfusion low-dose interleukin (IL)-2. Two patients were treated. While one patient had significant tumor regression and resultant clinical benefit, the other patient suffered a fatal histiocytic myocarditis. Therefore, this cohort was terminated for safety concerns. CONCLUSION: We describe a new and serious toxicity of immunotherapy from IFN-γ combined with HD Cy-based lymphodepletion and low-dose IL-2. While IFN-γ should not be used concurrently with HD Cy or with low dose IL-2, IFN-γ may still be important in sensitizing SS for ACT. Future studies should avoid using IFN-γ during the immediate period before/after cell infusion. TRIAL REGISTRATION NUMBERS: NCT04177021, NCT01957709, and NCT03063632.

Ginsenoside Rg1 protects against cigarette smoke-induced airway remodeling by suppressing the TGF-ß1/Smad3 signaling pathway.

Chronic obstructive pulmonary disease (COPD) is a devastating and common respiratory disease characterized by chronic inflammation and progressive airway remodeling. Ginsenoside Rg1 (GRg1), a major active component of Panax ginseng, has been found to possess beneficial properties against acute lung injury and respiratory diseases. However, the effects of GRg1 on airway remodeling in COPD remain unclear. In this study, we aimed to investigate the potential protective effects of GRg1 on airway remodeling induced by cigarette smoke (CS) and the underlying mechanism. A rat model of COPD was established in which the animals were subjected to CS and GRg1 daily for 12 weeks. Subsequently, we evaluated lung function, inflammatory responses, along with airway remodeling and associated signaling factors. GRg1 treatment was found to improve pulmonary function, reduce airway collagen volume fraction, and markedly reduce the expression of IL-6, TNF-α, α-SMA, and collagen I. Moreover, GRg1 treatment decreased the expression of TGF-ß1, TGF-ßR1, and phosphorylated-Smad3. In vitro, pretreatment of MRC5 human lung fibroblasts with GRg1 prior to exposure to cigarette smoke extract (CSE) reversed the cell ultrastructure disorder, decreased the expression of IL-6 and TNF-α, and significantly attenuated transdifferentiation of MRC5 cells by suppressing α-SMA and collagen I expression. Additionally, GRg1 suppressed the TGF-ß1/Smad3 signaling pathway in CSE-stimulated MRC5 cells, whereas Smad3 over-expression abolished the anti-transdifferentiation effect of GRg1. In conclusion, the results of our study demonstrated that GRg1 improves lung function and protects against CS-induced airway remodeling, in part by down-regulating the TGF-ß1/Smad3 signaling pathway.

Distinct Circulating Expression Profiles of Long Noncoding RNAs in Heart Failure Patients With Ischemic and Nonischemic Dilated Cardiomyopathy.

Ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM), with distinct long-term prognosis and responses to treatment, are two major problems that lead to heart failure (HF) ultimately. In this study, we investigated the long noncoding RNA (lncRNA) and messenger RNA (mRNA) expressions in the plasma of patients with DCM and ICM and analyzed the different lncRNA profile between the two groups. The microarray analysis identified 3,222 and 1,911 significantly differentially expressed lncRNAs and mRNAs between DCM and ICM group. The most enriched upregulated functional terms included positive regulation of I-kappaB kinase/nuclear factor-kappaB signaling and regulation of cellular localization, while the top 10 downregulated genes mainly consisted of acid secretion and myosin heavy chain binding. Furthermore, the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the differentially expressed lncRNA-coexpressed mRNAs between DCM and ICM group were significantly enriched in the natural killer cell mediated cytotoxicity and ras signaling pathway respectively. Quantitative real-time PCR confirmed 8 of 12 lncRNAs were upregulated in DCM group compared to ICM group which was consistent with the initial microarray results. The lncRNA/mRNA coexpression network indicated the possible functions of the validated lncRNAs. These findings revealed for the first time the specific expression pattern of both protein-coding RNAs and lncRNAs in plasma of HF patients due to DCM and ICM which may provide some important evidence to conveniently identify the etiology of myocardial dysfunctions and help to explore a better strategy for future HF prognosis evaluation.

Direct and indirect effects of liraglutide on hypothalamic POMC and NPY/AgRP neurons - Implications for energy balance and glucose control.

OBJECTIVE: The long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, stimulates insulin secretion and efficiently suppresses food intake to reduce body weight. As such, liraglutide is growing in popularity in the treatment of diabetes and chronic weight management. Within the brain, liraglutide has been shown to alter the activity of hypothalamic proopiomelanocortin (POMC) and Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons. Moreover, the acute activities of POMC and NPY neurons have been directly linked to feeding behavior, body weight, and glucose metabolism. Despite the increased usage of liraglutide and other GLP-1 analogues as diabetic and obesity interventions, the cellular mechanisms by which liraglutide alters the activity of metabolically relevant neuronal populations are poorly understood. METHODS: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to identify POMC and NPY neurons for patch-clamp electrophysiology experiments. RESULTS: We found that liraglutide directly activated arcuate POMC neurons via TrpC5 channels, sharing a similar mechanistic pathway to the adipose-derived peptide leptin. Liraglutide also indirectly increases excitatory tone to POMC neurons. In contrast, liraglutide inhibited NPY/AgRP neurons through post-synaptic GABAA receptors and enhanced activity of pre-synaptic GABAergic neurons, which required both TrpC5 subunits and K-ATP channels. In support of an additive role of leptin and liraglutide in suppressing food intake, leptin potentiated the acute effects of liraglutide to activate POMC neurons. TrpC5 subunits in POMC neurons were also required for the intact pharmacological effects of liraglutide on food intake and body weight. Thus, the current study adds to recent work from our group and others, which highlight potential mechanisms to amplify the effects of GLP-1 agonists in vivo. Moreover, these data highlight multiple sites of action (both pre- and post-synaptic) for GLP-1 agonists on this circuit. CONCLUSIONS: Taken together, our results identify critical molecular mechanisms linking GLP-1 analogues in arcuate POMC and NPY/AgRP neurons with metabolism.

The Therapeutic Role of Xenobiotic Nuclear Receptors Against Metabolic Syndrome.

BACKGROUND: Diabetes, with an increased prevalence and various progressive complications, has become a significant global health challenge. The concrete mechanisms responsible for the development of diabetes still remain incompletely unknown, although substantial researches have been conducted to search for the effective therapeutic targets. This review aims to reveal the novel roles of Xenobiotic Nuclear Receptors (XNRs), including the Peroxisome Proliferator-Activated Receptor (PPAR), the Farnesoid X Receptor (FXR), the Liver X Receptor (LXR), the Pregnane X Receptor (PXR) and the Constitutive Androstane Receptor (CAR), in the development of diabetes and provide potential strategies for research and treatment of metabolic diseases. METHODS: We retrieved a large number of original data about these five XNRs and organized to focus on their recently discovered functions in diabetes and its complications. RESULTS: Increasing evidences have suggested that PPAR, FXR, LXR ,PXR and CAR are involved in the development of diabetes and its complications through different mechanisms, including the regulation of glucose and lipid metabolism, insulin and inflammation response and related others. CONCLUSION: PPAR, FXR, LXR, PXR, and CAR, as the receptors for numerous natural or synthetic compounds, may be the most effective therapeutic targets in the treatment of metabolic diseases.

Combined IL-21-primed polyclonal CTL plus CTLA4 blockade controls refractory metastatic melanoma in a patient.

Adoptive transfer of peripheral blood-derived, melanoma-reactive CD8(+) cytotoxic T lymphocytes (CTLs) alone is generally insufficient to eliminate bulky tumors. Similarly, monotherapy with anti-CTLA4 infrequently yields sustained remissions in patients with metastatic melanoma. We postulated that a bolus of enhanced IL-21-primed polyclonal antigen-specific CTL combined with CTLA4 blockade might boost antitumor efficacy. In this first-in-human case study, the combination successfully led to a durable complete remission (CR) in a patient whose disease was refractory to both monoclonal CTL and anti-CTLA4. Long-term persistence and sustained anti-tumor activity of transferred CTL, as well as responses to nontargeted antigens, confirmed mutually beneficial effects of the combined treatment. In this first-in-human study, Chapuis et al. demonstrate that the combination of adoptive cellular therapy with CTLA4 blockade induces long-term remission in a melanoma patient resistant to both modalities administered serially and individually.

T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression.

Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy.

CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients.

BACKGROUND: T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. METHODS: We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS: The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. CONCLUSION: Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION: ClinicalTrials.gov NCT01865617. FUNDING: R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes.

Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We here describe an approach to identify single-chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by HLA molecules. We demonstrate that these scFvs can be successfully converted to full-length antibodies, termed MANAbodies, targeting "Mutation-Associated Neo-Antigens" bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for mutant peptides bound to predefined HLA types. In this way, we obtained two scFvs, one specific for a peptide encoded by a common KRAS mutant and the other by a common epidermal growth factor receptor (EGFR) mutant. The scFvs bound to these peptides only when the peptides were complexed with HLA-A2 (KRAS peptide) or HLA-A3 (EGFR peptide). We converted one scFv to a full-length antibody (MANAbody) and demonstrate that the MANAbody specifically reacts with mutant peptide-HLA complex even when the peptide differs by only one amino acid from the normal, WT form.