Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies.

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR-T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens.

Trends in baseline CD4 cell counts and risk factors for late antiretroviral therapy initiation among HIV-positive patients in Shanghai, a retrospective cross-sectional study.

BACKGROUNDS: There are few studies focus on the factors underlying the late initiation of ART in China. We analyzed the trends in the median CD4 cell counts among different patient groups over time and the risk factors for the late initiation of ART in Shanghai, China. METHODS: A retrospective cross-sectional survey was made in the Department of Infectious Disease of Shanghai Public Health Clinical Center which is a designated diagnosis and treatment center for HIV-positive patients in Shanghai during the period of January 1st, 2008--June 30th, 2014. Late ART initiation was defined as a CD4 cell count <200 cells/mm3 or having a clinical AIDS diagnosis prior to ART initiation. Trends in the median CD4 cell count at ART initiation and the proportion of late ART initiation by year were evaluated using Spearman's correlations and Chi-squared methods, respectively. We used a logistic regression model to analyze the risk factors for late ART initiation. The related factors collected in the multivariate model were the patient's age, gender, infection routes and marital status. RESULTS: A total of 3796 patients were analyzed in this study, with a median baseline CD4 cell count of 205 cells/mm3 [interquartile range: 75-287]. The median CD4 cell counts of patients initiating ART late increased from 76 cells/mm3 in 2008 to 103 cells/mm3 in 2014 (p < 0.001), and the proportion of late ART initiation decreased from 80% to 45% (p < 0.001). The risk factors for late ART initiation were male gender, heterosexual transmission and older age (>30 years) (p < 0.001). CONCLUSIONS: Notable improvements were made in the median CD4 cell count at ART initiation and the proportion of late ART initiation from 2008 to 2014. However, persons with high risk of HIV exposure who are male, older even heterosexual orientation should be given more opportunities to receive frequently screening, earlier diagnoses and timely treatment.

Targeted Delivery of an Anti-inflammatory PDE4 Inhibitor to Immune Cells via an Antibody-drug Conjugate.

Phosphodiesterase 4 (PDE4) inhibitors are approved for the treatment of some moderate to severe inflammatory conditions. However, dose-limiting side effects in the central nervous system and gastrointestinal tract, including nausea, emesis, headache, and diarrhea, have impeded the broader therapeutic application of PDE4 inhibitors. We sought to exploit the wealth of validation surrounding PDE4 inhibition by improving the therapeutic index through generation of an antibody-drug conjugate (ADC) that selectively targets immune cells through the CD11a antigen. The resulting ADC consisted of a human αCD11a antibody (based on efalizumab clone hu1124) conjugated to an analog of the highly potent PDE4 inhibitor GSK256066. Both the human αCD11a ADC and a mouse surrogate αCD11a ADC (based on the M17 clone) rapidly internalized into immune cells and suppressed lipololysaccharide (LPS)-induced TNFα secretion in primary human monocytes and mouse peritoneal cells, respectively. In a carrageenan-induced air pouch inflammation mouse model, treatment with the ADC significantly reduced inflammatory cytokine production in the air pouch exudate. Overall, these results provide compelling evidence for the feasibility of delivering drugs with anti-inflammatory activity selectively to the immune compartment via CD11a and the development of tissue-targeted PDE4 inhibitors as a promising therapeutic modality for treating inflammatory diseases.

LysZX4-NCA, a new endolysin with broad-spectrum antibacterial activity for topical treatment.

The prevalence of multidrug-resistant highly virulent Klebsiella pneumoniae (MDR-hvKP) requires the development of new therapeutic agents. Herein, a novel lytic phage vB_KpnS_ZX4 against MDR-hvKP was discovered in hospital sewage. Phage vB_KpnS_ZX4 had a short latent period (5 min) and a large burst size (230 PFU/cell). It can rapidly reduce the number of bacteria in vitro and improve survival rates of bacteremic mice in vivo from 0 to 80 % with a single injection of 108 PFU. LysZX4, an endolysin derived from vB_KpnS_ZX4, exhibits potent antimicrobial activity in vitro in combination with ethylenediaminetetraacetic acid (EDTA). The antimicrobial activity of LysZX4 was further enhanced by the fusion of KWKLFKI residues from cecropin A (LysZX4-NCA). In vitro antibacterial experiments showed that LysZX4-NCA exerts broad-spectrum antibacterial activity against clinical Gram-negative bacteria, including MDR-hvKP. Moreover, in the mouse model of MDR-hvKP skin infection, treatment with LysZX4-NCA resulted in a three-log reduction in bacterial burden on the skin compared to the control group. Therefore, the novel phages vB_KpnS_ZX4 and LysZX4-NCA are effective reagents for the treatment of systemic and local MDR-hvKP infections.

Development of a novel cholesterol tag-based system for trans-membrane transport of protein drugs.

The main technological difficulties of developing an intracellular (transmembrane) transport system for protein drugs lie in two points: i) overcoming the barriers in the cellular membrane, and ii) loading enough protein drugs, and particularly high-dose proteins, into particles. To address these two technological problems, we recently developed a novel cholesterol tag (C-Tag)-based transmembrane transport system. This pilot study found that the C-Tag dramatically improved the cellular uptake of Fab (902-fold, vs. Fab alone) into living cells, indicating that it successfully achieved transmembrane transport. Moreover, C-Tag-mediated membrane transport was verified using micron-scale large unilamellar vesicles (LUVs, approximately 1.5 µm)-based particles. The C-Tagged Fab was able to permeate the liposomal bilayer and it greatly enhanced (a 10.1-fold increase vs. Fab alone) internalization of proteins into the LUV-based particles, indicating that the C-Tag loaded enough proteins into particles for use of high-dose proteins. Accordingly, we established a novel C-Tag-based transport system that has overcome the known technological difficulties of protein transmembrane delivery, and this might be a useful technology for drug development in the future.

Use of folate-conjugated imaging agents to target alternatively activated macrophages in a murine model of asthma.

Pro-inflammatory macrophages play a prominent role in such autoimmune diseases as rheumatoid arthritis, Crohn's disease, psoriasis, sarcoidosis, and atherosclerosis. Because pro-inflammatory macrophages have also been shown to overexpress a receptor for the vitamin folic acid (i.e., folate receptor beta; FR-ß), folate-linked drugs have been explored for use in imaging and treatment of these same diseases. To determine whether allergic inflammatory disorders might be similarly targeted with folate-linked drugs, we have examined the characteristics of macrophages that are prominent in the pathogenesis of asthma. We report here that macrophages from the lungs of mice with experimental allergic asthma express FR-ß. We further document that these FR-ß(+) macrophages coexpress markers of alternatively activated (M2-type) macrophages, including the mannose receptor and arginase-1. Finally, we demonstrate that folate-conjugated fluorescent dyes and radioimaging agents can be specifically targeted to these asthmatic lung macrophages, with little uptake by macrophages present in healthy lung tissue. These data suggest strategies for the development of novel diagnostic agents for the imaging of asthma and other diseases involving alternatively activated macrophages.

Devising novel near-infrared aggregation-induced-emission luminogen labeling for point-of-care diagnosis of Mycobacterium tuberculosis.

Detecting and appropriately diagnosing a Mycobacterium tuberculosis infection remains technologically difficult because the pathogen commonly hides in macrophages in a dormant state. Described here is novel near-infrared aggregation-induced-emission luminogen (AIEgen) labeling developed by the current authors' laboratory for point-of-care (POC) diagnosis of an M. tuberculosis infection. The selectivity of AIEgen labeling, the labeling of intracellular M. tuberculosis by AIEgen, and the labeling of M. tuberculosis in sputum samples by AIEgen, along with its accuracy, sensitivity, and specificity, were preliminarily evaluated. Results indicated that this near-infrared AIEgen labeling had satisfactory selectivity and it labeled intracellular M. tuberculosis and M. tuberculosis in sputum samples. It had a satisfactory accuracy (95.7%), sensitivity (95.5%), and specificity (100%) for diagnosis of an M. tuberculosis infection in sputum samples. The current results indicated that near-infrared AIEgen labeling might be a promising novel diagnostic tool for POC diagnosis of M. tuberculosis infection, though further rigorous verification of these findings is required.

Sequelae of long COVID, known and unknown: A review of updated information.

Over three years have passed since the COVID-19 pandemic started. The dangerousness and impact of COVID-19 should definitely not be ignored or underestimated. Other than the symptoms of acute infection, the long-term symptoms associated with SARS-CoV-2 infection, which are referred to here as "sequelae of long COVID (LC)", are also a conspicuous global public health concern. Although such sequelae were well-documented, the understanding of and insights regarding LC-related sequelae remain inadequate due to the limitations of previous studies (the follow-up, methodological flaws, heterogeneity among studies, etc.). Notably, robust evidence regarding diagnosis and treatment of certain LC sequelae remain insufficient and has been a stumbling block to better management of these patients. This awkward situation motivated us to conduct this review. Here, we comprehensively reviewed the updated information, particularly focusing on clinical issues. We attempt to provide the latest information regarding LC-related sequelae by systematically reviewing the involvement of main organ systems. We also propose paths for future exploration based on available knowledge and the authors' clinical experience. We believe that these take-home messages will be helpful to gain insights into LC and ultimately benefit clinical practice in treating LC-related sequelae.

Characterization of a New Temperate Escherichia coli Phage vB_EcoP_ZX5 and Its Regulatory Protein.

The study of the interaction between temperate phages and bacteria is vital to understand their role in the development of human diseases. In this study, a novel temperate Escherichia coli phage, vB_EcoP_ZX5, with a genome size of 39,565 bp, was isolated from human fecal samples. It has a short tail and belongs to the genus Uetakevirus and the family Podoviridae. Phage vB_EcoP_ZX5 encodes three lysogeny-related proteins (ORF12, ORF21, and ORF4) and can be integrated into the 3'-end of guaA of its host E. coli YO1 for stable transmission to offspring bacteria. Phage vB_EcoP_ZX5 in lysogenized E. coli YO1+ was induced spontaneously, with a free phage titer of 107 PFU/mL. The integration of vB_EcoP_ZX5 had no significant effect on growth, biofilm, environmental stress response, antibiotic sensitivity, adherence to HeLa cells, and virulence of E. coli YO1. The ORF4 anti-repressor, ORF12 integrase, and ORF21 repressors that affect the lytic-lysogenic cycle of vB_EcoP_ZX5 were verified by protein overexpression. We could tell from changes of the number of total phages and the transcription level of phage genes that repressor protein is the key determinant of lytic-to-lysogenic conversion, and anti-repressor protein promotes the conversion from lysogenic cycle to lytic cycle.

Characterization of Novel Bacteriophage vB_KpnP_ZX1 and Its Depolymerases with Therapeutic Potential for K57 Klebsiella pneumoniae Infection.

A novel temperate phage vB_KpnP_ZX1 was isolated from hospital sewage samples using the clinically derived K57-type Klebsiella pneumoniae as a host. Phage vB_KpnP_ZX1, encoding three lysogen genes, the repressor, anti-repressor, and integrase, is the fourth phage of the genus Uetakevirus, family Podoviridae, ever discovered. Phage vB_KpnP_ZX1 did not show ideal bactericidal effect on K. pneumoniae 111-2, but TEM showed that the depolymerase Dep_ZX1 encoded on the short tail fiber protein has efficient capsule degradation activity. In vitro antibacterial results show that purified recombinant Dep_ZX1 can significantly prevent the formation of biofilm, degrade the formed biofilm, and improve the sensitivity of the bacteria in the biofilm to the antibiotics kanamycin, gentamicin, and streptomycin. Furthermore, the results of animal experiments show that 50 µg Dep_ZX1 can protect all K. pneumoniae 111-2-infected mice from death, whereas the control mice infected with the same dose of K. pneumoniae 111-2 all died. The degradation activity of Dep_ZX1 on capsular polysaccharide makes the bacteria weaken their resistance to immune cells, such as complement-mediated serum killing and phagocytosis, which are the key factors for its therapeutic action. In conclusion, Dep_ZX1 is a promising anti-virulence agent for the K57-type K. pneumoniae infection or biofilm diseases.

Artificial intelligence-assisted enumeration of ultra-small viruses with dual dark-field plasmon resonance probes.

Direct visual enumeration of viruses under dark-field microscope (DFM) using plasmon resonance probes (PRPs) is fast and convenient; however, it is greatly limited in the assay of real samples because of its inability to accurately identify false positives owing to non-specific adsorption. In this study, we propose an artificial intelligence (AI)-assisted DFM enumeration strategy for the accurate assay of Enterovirus A71 (an ultra-small human virus) using two PRPs; a 40 nm silver nanoparticle probe (SNP) that appears bright blue under DFM, and a 120 nm gold nanorod probe (GNP) that appears red under DFM. The capture chip was prepared by immobilizing the SNPs with antibodies on the glass to capture the target virus and to form dichromatic sandwich structures with the GNPs, followed by imaging under a dark field (DF). Subsequently, the DF images of the capture chip were subjected to a two-step screening: first, using image processing, and thereafter using the AI algorithm screening to eliminate false positive results and background noise. The results revealed that the data from the AI-assisted dual PRPs assay were highly consistent with those of quantitative PCR (qPCR), and that the sensitivity with a minimum detectable concentration of 3 copies/µL was 5 times higher than that of qPCR. The entire analysis was completed within 45 min. Therefore, our AI-assisted virus enumeration strategy with two DF PRPs holds great potential for ultra-sensitive and accurate quantification of viruses in real samples.

Evaluation of the reducing potential of PSMA-containing endosomes by FRET imaging.

Aim: Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in healthy tissues. For many ligand-targeted drug conjugates, a critical aspect of conjugate design lies in engineering release of the therapeutic payload to occur only after its internalization by targeted cells. Because disulfide bond reduction is frequently exploited to ensure intracellular drug release, an understanding of the redox properties of endocytic compartments can be critical to ligand-targeted drug design. While the redox properties of folate receptor trafficking endosomes have been previously reported, little is known about the trafficking of prostate-specific membrane antigen (PSMA), a receptor that is experiencing increasing use for drug targeting in humans. Methods: To obtain this information, we have constructed a PSMA-targeted fluorescence resonance energy transfer pair that reports on disulfide bond reduction by changing fluorescence from red to green. Results: We show here that this reporter exhibits rapid and selective uptake by PSMA-positive cells, and that reduction of its disulfide bond proceeds steadily but incompletely following internalization. The fact that maximal disulfide reduction reaches only ~50%, even after 24 h incubation, suggests that roughly half of the conjugates must traffic through endosomes that display no reducing capacity. Conclusion: As the level of disulfide reduction differs between PSMA trafficked and previously published folate trafficked conjugates, it also follows that not all internalizing receptors are translocated through similar intracellular compartments. Taken together, these data suggest that the efficiency of disulfide bond reduction must be independently analyzed for each receptor trafficking pathway when disulfide bond reduction is exploited for intracellular drug release.

Heat shock protein 90 (Hsp90) regulates the stability of transforming growth factor beta-activated kinase 1 (TAK1) in interleukin-1beta-induced cell signaling.

Heat shock protein 90 (Hsp90) is an abundantly and ubiquitously expressed chaperone with majority of client proteins which act as signal molecules. Transforming growth factor beta-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK), and is essential in interleukin-1beta (IL-1beta) triggered signaling pathways. In the present study, we found that Hsp90 plays an important role in regulating IL-1beta signaling by keeping TAK1 stability. The results showed that the specific inhibitor geldanamycin (GA) of Hsp90 dramatically inhibited IL-1beta stimulated TAK1-MAPKs and TAK1-nuclear factor-kappaB (NF-kappaB) activation, resulting in the decrease of cyclooxygenase-2 (COX-2) protein expression. Silencing Hsp90 expression through RNA interference (RNAi) also down-regulated TAK1, as well as attenuated IL-1beta induced phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38 MAPKs, and degradation of IkappaBalpha. The same results were obtained in T6RZC stable cells which initiated IL-1beta-induced cell signaling at the level of the oligomerization and ubquitination of TNF receptor-associated factor 6 (TRAF6). We further found that Hsp90 formed a complex with TAK1 via its N-terminal domain and GA destabilized TAK1 and induced TAK1 degradation through proteasome pathway. Taken together our results demonstrate that Hsp90 regulates IL-1beta-induced signaling by interacting with TAK1 and maintaining the stability of TAK1, suggesting that Hsp90 might act as the chaperone of TAK1 in immune and inflammatory responses related with IL-1 signal cascades.

Recombinant protein glutathione S-transferases P1 attenuates inflammation in mice.

We have reported that intracellular glutathione S-transferases P1 (GSTP1) suppresses LPS (lipopolysaccharide)-induced excessive production of pro-inflammatory factors by inhibiting LPS-stimulated MAPKs (mitogen-activated protein kinases) as well as NF-kappaB activation. But under pathogenic circumstances, physiologic levels of GSTP1 are insufficient to stem pro-inflammatory signaling. Here we show that LPS-induced up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW246.7 cells is significantly reduced by incubating cells with recombinant GSTP1 protein. In vivo study demonstrates that treatment of mice (i.p.) with recombinant GSTP1 protein effectively suppresses the devastating effects of acute inflammation, which includes reduction of mortality rate of endotoxic shock, alleviation of LPS-induced acute lung injury and abrogation of thioglycolate (TG)-induced peritoneal deposition of leukocytes and polymorphonuclear cells (PMNs). Meanwhile, GSTP1 prevented LPS-induced TNF-alpha, IL-1beta, MCP-1 and NO production. Further investigation by using confocal microscopy and flow cytometry shows that recombinant GSTP1 protein can be delivered into RAW246.7 cells, mouse peritoneal macrophages and HEK 293 cells suggesting that extracellular GSTP1 protein could be transported across plasma membrane and act as a cytosolic protein. In conclusion our research demonstrates a new finding that increasing cellular GSTP1 level by supplement of recombinant GSTP1 effectively suppresses the devastating effects of acute inflammation.

Dual-Enzyme Crosslinking and Post-polymerization for Printing of Polysaccharide-Polymer Hydrogel.

Polymer hydrogels are ideal bioprinting scaffolds for cell-loading and tissue engineering due to their extracellular-matrix-like structure. However, polymer hydrogels that are easily printed tend to have poor strength and fragile properties. The gradually polymerized reinforcement after hydrogel printing is a good method to solve the contradiction between conveniently printed and high mechanical strength requirement. Here, a new succinct approach has been developed to fabricate the printable composite hydrogels with tunable strength. We employed the HRP@GOx dual enzyme system to initiate the immediate crosslinking of chondroitin sulfate grafted with tyrosine and the gradual polymerization of monomers to form the composite hydrogels. The detailed two-step gelation mechanism was confirmed by the Fluorescence spectroscopy, Electron paramagnetic resonance spectroscopy and Gel permeation chromatography, respectively. The final composite hydrogel combines the merits of enzymatic crosslinking hydrogels and polymerized hydrogels to achieve adjustable mechanical strength and facile printing performance. The dual-enzyme regulated polymer composite hydrogels are the promising bioscaffolds as organoid, implanted materials, and other biomedical applications.

Depletion of activated macrophages with a folate receptor-beta-specific antibody improves symptoms in mouse models of rheumatoid arthritis.

OBJECTIVES: Most therapies for autoimmune and inflammatory diseases either neutralize or suppress production of inflammatory cytokines produced by activated macrophages (e.g., TNFα, IL-1, IL-6, IL-17, GM-CSF). However, no approved therapies directly target this activated subset of macrophages. METHODS: First, we undertook to examine whether the folate receptor beta (FR-ß) positive subpopulation of macrophages, which marks the inflammatory subset in animal models of rheumatoid arthritis, might constitute the prominent population of macrophages in inflamed lesions in humans. Next, we utilized anti-FR-ß monoclonal antibodies capable of mediating antibody-dependent cell cytotoxicity (ADCC) to treat animal models of rheumatoid arthritis and peritonitis. RESULTS: Human tissue samples of rheumatoid arthritis, Crohn's disease, ulcerative colitis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, chronic obstructive pulmonary disease, systemic lupus erythematosus, psoriasis, and scleroderma are all characterized by dramatic accumulation of macrophages that express FR-ß, a protein not expressed on resting macrophages or any other healthy tissues. A monoclonal antibody to FR-ß accumulates specifically in inflamed lesions of murine inflammatory disease models and successfully treats such models of rheumatoid arthritis and peritonitis. More importantly, elimination of FR-ß-positive macrophages upon treatment with an anti-FR-ß monoclonal antibody promotes the departure of other immune cells, including T cells, B cells, neutrophils, and dendritic cells from the inflamed lesions. CONCLUSIONS: These data suggest that specific elimination of FR-ß-expressing macrophages may constitute a highly specific therapy for multiple autoimmune and inflammatory diseases and that a recently developed human anti-human FR-ß monoclonal antibody (m909) might contribute to suppression of this subpopulation of macrophages.

Assessment of folate receptor alpha and beta expression in selection of lung and pancreatic cancer patients for receptor targeted therapies.

A number of folate receptor (FR) targeted small molecular drugs and monoclonal antibodies have been introduced into clinical trials to treat FR positive cancers. Because the therapeutic efficacy of these drugs depends prominently on the level of FR-α expression on the cancer cells, patients have been commonly selected for FR-targeted therapies based on the intensity of a folate-targeted radioimaging agent. Unfortunately, uptake of such imaging agents can be mediated by both major isoforms of the folate receptor, FR-α and FR-ß. Logically, if the FR positive cell population in a tumor mass is dominated by FR-ß positive macrophages, patients could be selected for therapy that have few FR-expressing cancer cells. Although several IHC studies have examined expression of either FR-α or FR-ß, no study to date has investigated expression of both FR-α and FR-ß in the same tumor mass. Herein, we utilize monoclonal antibodies specific for FR-α (mAb343) and FR-ß (m909) to query each isoform's expression in a range of cancers. We show that lung and pancreatic adenocarcinomas express the full spectrum of FR-α and FR-ß combinations with ~76% of lung adenocarcinomas expressing both FR-α and FR-ß while pancreatic cancers express primarily FR-ß. Thus, while folate-targeted imaging of lung cancer patients might accurately reflect the expression of FR-α on lung cancer cells, imaging of pancreatic cancer patients could mislead a physician into treating a nonresponding patient. Overall, these data suggest that an independent analysis of both FR-α and FR-ß should be obtained to predict the potential efficacy of a folate-targeted drug.

Modulations of Keap1-Nrf2 signaling axis by TIIA ameliorated the oxidative stress-induced myocardial apoptosis.

Mounting evidence has strongly implicated oxidative stress in the development of cardiac dysfunction, and myocardial apoptosis contributes to the pathogenesis of heart failure. Quantitative cardiac proteomics data revealed that pressure load by TAC resulted in a significant decline in mitochondrial metabolic activity, where TIIA (Tanshinone IIA sulfonate) treatment reversed it in vivo, which might be mediated by Nrf2. In NRVMs, TIIA treatment ameliorated H2O2-induced caspase-3/9 activations through the suppression of p38 and mTOR signaling pathways, where caspase-mediated cleavage of YY1 and PARP resulted in the defects in mitochondrial biogenesis and DNA repair, and this event finally led to cardiomyocyte apoptosis. Mass spectrometry analysis showed that TIIA hydrophobically interacted with Keap1 (the cytoplasmic repressor of Nrf2) and induced its degradation in vitro. Site-directed mutagenesis of Keap1 identified V122/V123/I125 to be the critical residues for the TIIA-induced de-dimerization and degradation of Keap1. Besides, TIIA treatment also epigenetically up-regulated Nrf2 gene transcription, where it hypomethylated the first 5 CpGs of Nrf2 promoter. Furthermore, cardiac-specific Nrf2 knockout mice exhibited the significantly dampened anti-apoptotic effects of TIIA.

Expression of functional folate receptors in multiple myeloma.

Receptor-targeted delivery of imaging and therapeutic agents has emerged as an attractive strategy to diagnosis and treat many diseases including cancer. One of the most well-studied receptors for targeted therapies is the folate receptor (FR) family. FR-α and FR-ß are present on many cancers with little expression in normal tissues; leading to the testing of at least six folate-targeted drugs in human clinical trials for various cancers. However, the expression of FR in blood cancers has not been fully explored with no reports of FR expression in myelomas. Herein, we report the expression of both FR-α and FR-ß on CD138 + plasma cells isolated from patients with multiple myeloma. In addition, all-trans retinoic acid was shown to increase the levels of FR-α and FR-ß in two myeloma cell lines. Altogether, this data suggests that folate-targeted therapies for the treatment of multiple myeloma warrants further investigation.

Bi-specific ligand-controlled chimeric antigen receptor T-cell therapy for non-small cell lung cancer.

Our goal is to develop a switch-controlled approach to enable better control of reactivity and safety of chimeric antigen receptor (CAR)-T therapy for non-small-cell lung cancer (NSCLC). Lentiviral transduction was performed to generate anti-FITC CAR-T cells and target cells stably expressing either isoform of the folate receptor. Colorimetric-based cytotoxic assay, enzyme-linked immunosorbent assay, and multiparametric flow cytometry analysis were used to evaluate the specificity and activity of CAR-T cells in vitro. Human primary T cells stably expressing the fully human anti-FITC CAR were generated. Anti-FITC CAR-T cells displayed antigen-specific and folate-FTIC dependent reactivity against engineered A549-FRα and THP-1-FRß. The selective activation and proliferation of anti-FITC CAR-T cells in vitro stringently relied on the co-existence of folate-FITC and FR- expressing target cells and was dose-titratable with the folate-FITC switch. The excellent in vitro efficacy and specificity of an adaptor-controlled CAR-T therapy to target both tumor cells and tumor-associated macrophages in NSCLCs were validated.