Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2.

SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR ) antibodies. VNAR s are small (<15 kDa) and can reach deep into the pockets or grooves of the target antigen. Here, we have isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naïve nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other ß coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens.

Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice.

BACKGROUND AND AIMS: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties. METHODS: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgcnull (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgcnull (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice. RESULTS: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active ß-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. CONCLUSIONS: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.

MicroRNA-1291-5p Sensitizes Pancreatic Carcinoma Cells to Arginine Deprivation and Chemotherapy through the Regulation of Arginolysis and Glycolysis.

Cancer cells are dysregulated and addicted to continuous supply and metabolism of nutritional glucose and amino acids (e.g., arginine) to drive the synthesis of critical macromolecules for uncontrolled growth. Recent studies have revealed that genome-derived microRNA (miRNA or miR)-1291-5p (miR-1291-5p or miR-1291) may modulate the expression of argininosuccinate synthase (ASS1) and glucose transporter protein type 1 (GLUT1). We also developed a novel approach to produce recombinant miR-1291 agents for research, which are distinguished from conventional chemo-engineered miRNA mimics. Herein, we firstly demonstrated that bioengineered miR-1291 agent was selectively processed to high levels of target miR-1291-5p in human pancreatic cancer (PC) cells. After the suppression of ASS1 protein levels, miR-1291 perturbed arginine homeostasis and preferably sensitized ASS1-abundant L3.3 cells to arginine deprivation therapy. In addition, miR-1291 treatment reduced the protein levels of GLUT1 in both AsPC-1 and PANC-1 cells, leading to a lower glucose uptake (deceased > 40%) and glycolysis capacity (reduced approximately 50%). As a result, miR-1291 largely improved cisplatin efficacy in the inhibition of PC cell viability. Our results demonstrated that miR-1291 was effective to sensitize PC cells to arginine deprivation treatment and chemotherapy through targeting ASS1- and GLUT1-mediated arginolysis and glycolysis, respectively, which may provide insights into understanding miRNA signaling underlying cancer cell metabolism and development of new strategies for the treatment of lethal PC. SIGNIFICANCE STATEMENT: Many anticancer drugs in clinical use and under investigation exert pharmacological effects or improve efficacy of coadministered medications by targeting cancer cell metabolism. Using new recombinant miR-1291 agent, we revealed that miR-1291 acts as a metabolism modulator in pancreatic carcinoma cells through the regulation of argininosuccinate synthase- and glucose transporter protein type 1-mediated arginolysis and glycolysis. Consequently, miR-1291 effectively enhanced the efficacy of arginine deprivation (pegylated arginine deiminase) and chemotherapy (cisplatin), offering new insights into development of rational combination therapies.

Histone methyltransferase NSD2 mediates the survival and invasion of triple-negative breast cancer cells via stimulating ADAM9-EGFR-AKT signaling.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with a poor prognosis due to the lack of an effective targeted therapy. Histone lysine methyltransferases (KMTs) have emerged as attractive drug targets for cancer therapy. However, the function of the majority of KMTs in TNBC has remained largely unknown. In the current study, we found that KMT nuclear receptor binding SET domain protein 2 (NSD2) is overexpressed in TNBC tumors and that its overexpression is associated with poor survival of TNBC patients. NSD2 regulates TNBC cell survival and invasion and is required for tumorigenesis and tumor growth. Mechanistically, NSD2 directly controls the expression of EGFR and ADAM9, a member of the ADAM (a disintegrin and metalloproteinase) family that mediates the release of growth factors, such as HB-EGF. Through its methylase activity, NSD2 overexpression stimulates EGFR-AKT signaling and promotes TNBC cell resistance to the EGFR inhibitor gefitinib. Together, our results identify NSD2 as a major epigenetic regulator in TNBC and provide a rationale for targeting NSD2 alone or in combination with EGFR inhibitors as a targeted therapy for TNBC.

Bioengineered Noncoding RNAs Selectively Change Cellular miRNome Profiles for Cancer Therapy.

Noncoding RNAs (ncRNAs) produced in live cells may better reflect intracellular ncRNAs for research and therapy. Attempts were made to produce biologic ncRNAs, but at low yield or success rate. Here we first report a new ncRNA bioengineering technology using more stable ncRNA carrier (nCAR) containing a pre-miR-34a derivative identified by rational design and experimental validation. This approach offered a remarkable higher level expression (40%-80% of total RNAs) of recombinant ncRNAs in bacteria and gave an 80% success rate (33 of 42 ncRNAs). New FPLC and spin-column based methods were also developed for large- and small-scale purification of milligrams and micrograms of recombinant ncRNAs from half liter and milliliters of bacterial culture, respectively. We then used two bioengineered nCAR/miRNAs to demonstrate the selective release of target miRNAs into human cells, which were revealed to be Dicer dependent (miR-34a-5p) or independent (miR-124a-3p), and subsequent changes of miRNome and transcriptome profiles. miRNA enrichment analyses of altered transcriptome confirmed the specificity of nCAR/miRNAs in target gene regulation. Furthermore, nCAR assembled miR-34a-5p and miR-124-3p were active in suppressing human lung carcinoma cell proliferation through modulation of target gene expression (e.g., cMET and CDK6 for miR-34a-5p; STAT3 and ABCC4 for miR-124-3p). In addition, bioengineered miRNA molecules were effective in controlling metastatic lung xenograft progression, as demonstrated by live animal and ex vivo lung tissue bioluminescent imaging as well as histopathological examination. This novel ncRNA bioengineering platform can be easily adapted to produce various ncRNA molecules, and biologic ncRNAs hold the promise as new cancer therapeutics.

Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells.

The nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor in the regulation of many oxidative enzymes and efflux transporters critical for oxidative stress and cellular defense against xenobiotics. NRF2 is dysregulated in patient osteosarcoma (OS) tissues and correlates with therapeutic outcomes. Nevertheless, research on the NRF2 regulatory pathways and its potential as a therapeutic target is limited to the use of synthetic small interfering RNA (siRNA) carrying extensive artificial modifications. Herein, we report successful high-level expression of recombinant siRNA against NRF2 in Escherichia coli using our newly established noncoding RNA bioengineering technology, which was purified to >99% homogeneity using an anion-exchange fast protein liquid chromatography method. Bioengineered NRF2-siRNA was able to significantly knock down NRF2 mRNA and protein levels in human OS 143B and MG63 cells, and subsequently suppressed the expression of NRF2-regulated oxidative enzymes [heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1] and elevated intracellular levels of reactive oxygen species. In addition, recombinant NRF2-siRNA was effective to sensitize both 143B and MG63 cells to doxorubicin, cisplatin, and sorafenib, which was associated with significant downregulation of NRF2-targeted ATP-binding cassette (ABC) efflux transporters (ABCC3, ABCC4, and ABCG2). These findings support that targeting NRF2 signaling pathways may improve the sensitivity of cancer cells to chemotherapy, and bioengineered siRNA molecules should be added to current tools for related research.

LDL Receptor-Related Protein 6 Modulates Ret Proto-Oncogene Signaling in Renal Development and Cystic Dysplasia.

Hypoplastic and/or cystic kidneys have been found in both LDL receptor-related protein 6 (Lrp6)- and ß-catenin-mutant mouse embryos, and these proteins are key molecules for Wnt signaling. However, the underlying mechanisms of Lrp6/ß-catenin signaling in renal development and cystic formation remain poorly understood. In this study, we found evidence that diminished cell proliferation and increased apoptosis occur before cystic dysplasia in the renal primordia of Lrp6-deficient mouse embryos. The expression of Ret proto-oncogene (Ret), a critical receptor for the growth factor glial cell line-derived neurotrophic factor (GDNF), which is required for early nephrogenesis, was dramatically diminished in the mutant renal primordia. The activities of other representative nephrogenic genes, including Lim1, Pax2, Pax8, GDNF, and Wnt11, were subsequently diminished in the mutant renal primordia. Molecular biology experiments demonstrated that Ret is a novel transcriptional target of Wnt/ß-catenin signaling. Wnt agonist lithium promoted Ret expression in vitro and in vivo. Furthermore, Lrp6-knockdown or lithium treatment in vitro led to downregulation or upregulation, respectively, of the phosphorylated mitogen-activated protein kinases 1 and 3, which act downstream of GDNF/Ret signaling. Mice with single and double mutations of Lrp6 and Ret were perinatal lethal and demonstrated gene dosage-dependent effects on the severity of renal hypoplasia during embryogenesis. Taken together, these results suggest that Lrp6-mediated Wnt/ß-catenin signaling modulates or interacts with a signaling network consisting of Ret cascades and related nephrogenic factors for renal development, and the disruption of these genes or signaling activities may cause a spectrum of hypoplastic and cystic kidney disorders.

Multi-scale object detection in UAV images based on adaptive feature fusion.

With the widespread use of UAVs, UAV aerial image target detection technology can be used for practical applications in the military, traffic planning, personnel search and rescue and other fields. In this paper, we propose a multi-scale UAV aerial image detection method based on adaptive feature fusion for solving the problem of detecting small target objects in UAV aerial images. This method automatically adjusts the convolution kernel receptive field and reduces the redundant background of the image by adding an adaptive feature extraction module (AFEM) to the backbone network. This enables it to obtain more accurately and effectively small target feature information. In addition, we design an adaptive feature weighted fusion network (SBiFPN) to effectively enhance the representation of shallow feature information of small targets. Finally, we add an additional small target detection scale to the original network to expand the receptive field of the network and strengthen the detection of small target objects. The training and testing are carried out on the VisDrone public dataset. The experimental results show that the proposed method can achieve 38.5% mAP, which is 2.0% higher than the baseline network YOLOv5s, and can still detect the UAV aerial image well in complex scenes.

Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability.

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Developmental and androgenic regulation of chromatin regulators EZH2 and ANCCA/ATAD2 in the prostate Via MLL histone methylase complex.

BACKGROUND: Chromatin regulators ANCCA and EZH2 are overexpressed in prostate cancer and play crucial roles in androgen-stimulated and castration-refractory prostate tumor growth and survival. However, how their expression is regulated in the tumors and whether they play a role in prostate development remains unclear. METHODS: Prostate tissue from different developmental stages of mouse and human were examined by IHC, qRT-PCR and Western for expression of ANCCA, EZH2, and Ki-67. Animals were castrated and T-implanted for the expression response in normal prostate and tumors. siRNA knockdown and ChIP were performed for the mechanism of ANCCA regulation of EZH2. RESULTS: In contrast to their very low level expression in adult prostate, ANCCA and EZH2 are strongly expressed in the epithelium and mesenchyme of mouse and human UGS. Their expression becomes more restricted to epithelial cells during later development and displays a second peak during puberty, which correlates with the proliferative status of the epithelium. Importantly, their expression in normal prostate and tumors is strongly suppressed by castration and markedly induced by testosterone replacement. While androgen suppresses EZH2 in CRPC cells, in LNCaP cells, physiological concentrations of androgen stimulate expression of PRC2 genes (EZH2, SUZ12, and EED), which is mediated by androgen-induced ANCCA and involves E2F and histone H3K4me3 methylase MLL1 complex. CONCLUSION: EZH2 and ANCCA are androgen regulated and strongly expressed in early prostate morphogenesis and during puberty, suggesting their important role in prostate development. Regulation of EZH2 by ANCCA emphasizes bromodomain protein ANCCA as a potential therapeutic target against prostate cancer.

Human VH-based chimeric antigen receptor T cells targeting glypican 3 eliminate tumors in preclinical models of HCC.

BACKGROUND AND AIMS: Efficacy of chimeric antigen receptor (CAR) T cells for treating solid tumors, including HCC, remains a challenge. Nanobodies are emerging building blocks of CAR T cells due to their small size and high expression. Membrane proximal sites have been shown as attractive epitopes of CAR T cells. However, current CAR formats are not tailored toward nanobodies or targeting membrane distal epitopes. APPROACH AND RESULTS: Using hYP7 Fv (membrane proximal) and HN3 VH nanobody (membrane distal) as GPC3 targeting elements, we sought to determine how hinges and transmembrane portions of varying structures and sizes affect CAR T-cell function. We generated multiple permutations of CAR T cells containing CD8, CD28, IgG4, and Fc domains. We show that engineered HN3 CAR T cells can be improved by 2 independent, synergistic changes in the hinge and transmembrane domains. The T cells expressing the HN3 CAR which contains the hinge region of IgG4 and the CD28 transmembrane domain (HN3-IgG4H-CD28TM) exhibited high cytotoxic activity and caused complete HCC tumor eradication in immunodeficient mice. HN3-IgG4H-CD28TM CAR T cells were enriched for cytotoxic-memory CD8+ T cells and NFAT signals, and reduced ß catenin levels in HCC cells. CONCLUSION: Our findings indicate that altering the hinge and transmembrane domains of a nanobody-based CAR targeting a distal GPC3 epitope, in contrast to a membrane proximal epitope, lead to robust T-cell signaling and induce swift and durable eradication of HCC tumors.

Individual exposure of ambient particulate matters and eosinophilic chronic rhinosinusitis with nasal Polyps: Dose-Response, mediation effects and recurrence prediction.

PURPOSE: We evaluated the association between ambient particulate matter (PM) exposure and eosinophilic chronic rhinosinusitis with nasal polyps (CRSwNP), and predicted the CRSwNP recurrence risk using machine learning algorithms. METHODS: In total, 1,086 patients with CRSwNP were recruited from nine hospitals in China during 2014-2019. The average annual concentrations of ambient PMs before surgery were assessed using satellite-based daily concentrations of PM2.5 and PM10 for a 1 × 1-km2 area. Linear regression and logistic regression models were used to evaluate the associations of PM exposure with eosinophilia and risks of eosinophilic CRSwNPs. In addition, mediation effect analysis was used to validate the interrelationships of the aforementioned factors. Finally, machine learning algorithms were used to predict the recurrence risks of CRSwNPs. RESULTS: There was a significantly increased risk of eosinophilic CRSwNPs with each 10 µg/m3 increase in PMs, with odds ratios (ORs) of 1.039 (95% confidence interval [CI] = 1.007-1.073) for PM10 and 1.058 (95% CI = 1.007- 1.112) for PM2.5. Eosinophils had a significant mediation effect, which accounted for 52% and 35% of the relationships of CRSwNP recurrence with PM10 and PM2.5, respectively. Finally, we developed a naïve Bayesian model to predict the risk of CRSwNP recurrence based on PM exposure, inflammatory data, and patients' demographic factors. CONCLUSIONS: Increased PM exposure is associated with an increased risk of eosinophilic CRSwNP in China. Therefore, patients with eosinophilic CRSwNP should reduce PM exposure to mitigate its harmful impacts.

Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3-Expressing Liver Cancer by Immuno-PET.

Primary liver cancer is the third leading cause of cancer-related deaths, and its incidence and mortality are increasing worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of primary liver cancer cases. Glypican-3 (GPC3) is a heparan sulfate proteoglycan that histopathologically defines HCC and represents an attractive tumor-selective marker for radiopharmaceutical imaging and therapy for this disease. Single-domain antibodies are a promising scaffold for imaging because of their favorable pharmacokinetic properties, good tumor penetration, and renal clearance. Although conventional lysine-directed bioconjugation can be used to yield conjugates for radiolabeling full-length antibodies, this stochastic approach risks negatively affecting target binding of the smaller single-domain antibodies. To address this challenge, site-specific approaches have been explored. Here, we used conventional and sortase-based site-specific conjugation methods to engineer GPC3-specific human single-domain antibody (HN3) PET probes. Methods: Bifunctional deferoxamine (DFO) isothiocyanate was used to synthesize native HN3 (nHN3)-DFO. Site-specifically modified HN3 (ssHN3)-DFO was engineered using sortase-mediated conjugation of triglycine-DFO chelator and HN3 containing an LPETG C-terminal tag. Both conjugates were radiolabeled with 89Zr, and their binding affinity in vitro and target engagement of GPC3-positive (GPC3+) tumors in vivo were determined. Results: Both 89Zr-ssHN3 and 89Zr-nHN3 displayed nanomolar affinity for GPC3 in vitro. Biodistribution and PET/CT image analysis in mice bearing isogenic A431 and A431-GPC3+ xenografts, as well as in HepG2 liver cancer xenografts, showed that both conjugates specifically identify GPC3+ tumors. 89Zr-ssHN3 exhibited more favorable biodistribution and pharmacokinetic properties, including higher tumor uptake and lower liver accumulation. Comparative PET/CT studies on mice imaged with both 18F-FDG and 89Zr-ssHN3 showed more consistent tumor accumulation for the single-domain antibody conjugate, further establishing its potential for PET imaging. Conclusion: 89Zr-ssHN3 showed clear advantages in tumor uptake and tumor-to-liver signal ratio over the conventionally modified 89Zr-nHN3 in xenograft models. Our results establish the potential of HN3-based single-domain antibody probes for GPC3-directed PET imaging of liver cancers.

A 5' extended IFN-stimulating response element is crucial for IFN-gamma-induced tripartite motif 22 expression via interaction with IFN regulatory factor-1.

Interferon-gamma is crucial for the noncytopathic clearance of hepatitis B virus. In our previous study, we demonstrated that an IFN-gamma-inducible molecule, tripartite motif (TRIM) 22, played an important role in antiviral immunity against hepatitis B virus. However, the molecular mechanism of TRIM22 induction by IFN-gamma is still unclear. In this study, we identified a novel cis-element termed 5' extended IFN-stimulating response element (5' eISRE) that was crucial for IFN-gamma inducibility of TRIM22 through transfection assays with luciferase reporter constructs and EMSAs. The 5' eISRE consists of an ISRE-like motif (ACTTTCGTTTCTC) and a 6-bp sequence (AATTTA) upstream of it, and all three thymine triplets of this cis-element (AATTTAACTTTCGTTTCTC) were revealed to contribute to the IFN-gamma inducibility of TRIM22 by site-directed mutagenesis. Further studies showed that upon IFN-gamma stimulation, the 5' eISRE could be bound by IFN regulatory factor-1 (IRF-1), but not by STAT1, as demonstrated by supershift analysis and an ELISA-based transcription factor assay. Moreover, overexpression of IRF-1 significantly induced TRIM22 expression, whereas silencing of IRF-1 with specific short interference RNA abolished IFN-gamma-induced TRIM22 expression in HepG2 cells, indicating an IRF-1-dependent expression of TRIM22. Taken together, it was demonstrated in this study that a novel cis-element, 5' eISRE, was crucial for the IFN-gamma-induced transcriptional activity of the TRIM22 gene via interaction with IRF-1.

Production and Purification of Shark and Camel Single-Domain Antibodies from Bacterial and Mammalian Cell Expression Systems.

Single-domain antibodies, including the antigen-binding variable domains of the shark immunoglobulin new antigen receptor and the camelid variable region of the heavy chain, are the smallest antigen recognition domains (∼15 kDa) and have unique characteristics compared to conventional antibodies. They are capable of binding epitopes that are hard to access for classical antibodies and can also be used for therapeutics or diagnostics or as modular building blocks for multi-domain constructs, antibody-drug conjugates, immunotoxins, or chimeric antigen receptor therapy. This article contains detailed procedures for the purification and validation of two single-domain antibodies (one shark and one camel), which bind to the S2 subunit of the SARS-CoV-2 spike protein, using both bacterial and mammalian cell expression systems. It provides a comprehensive reference for the production of single-domain antibodies with high yield, good quality, and purity. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Production of single-domain antibodies from Escherichia coli Alternate Protocol: Production of single-domain antibodies using the mammalian cell line Expi293F Support Protocol 1: Production and purification of single-domain antibodies on a small scale with the polymyxin B method Support Protocol 2: Validation of single-domain antibodies by ELISA.

Nuclear TIGAR mediates an epigenetic and metabolic autoregulatory loop via NRF2 in cancer therapeutic resistance.

Metabolic and epigenetic reprogramming play important roles in cancer therapeutic resistance. However, their interplays are poorly understood. We report here that elevated TIGAR (TP53-induced glycolysis and apoptosis regulator), an antioxidant and glucose metabolic regulator and a target of oncogenic histone methyltransferase NSD2 (nuclear receptor binding SET domain protein 2), is mainly localized in the nucleus of therapeutic resistant tumor cells where it stimulates NSD2 expression and elevates global H3K36me2 mark. Mechanistically, TIGAR directly interacts with the antioxidant master regulator NRF2 and facilitates chromatin recruitment of NRF2, H3K4me3 methylase MLL1 and elongating Pol-II to stimulate the expression of both new (NSD2) and established (NQO1/2, PRDX1 and GSTM4) targets of NRF2, independent of its enzymatic activity. Nuclear TIGAR confers cancer cell resistance to chemotherapy and hormonal therapy in vitro and in tumors through effective maintenance of redox homeostasis. In addition, nuclear accumulation of TIGAR is positively associated with NSD2 expression in clinical tumors and strongly correlated with poor survival. These findings define a nuclear TIGAR-mediated epigenetic autoregulatory loop in redox rebalance for tumor therapeutic resistance.

Identification of tripartite motif-containing 22 (TRIM22) as a novel NF-κB activator.

Increasing evidence suggests that TRIM family proteins may play important roles in the regulation of innate immune signaling pathways. Here we report TRIM22 is involved in the activation of NF-κB. It was found that overexpression of TRIM22 could dose-dependently activate NF-κB as demonstrated by reporter gene assay and electrophoretic mobility shift assay, but had no effect on the activity of other transcription factors, including NF-AT, AP-1, C/EBP and IRFs. Further study showed that both the N-terminal RING domain and C-terminal SPRY domain were crucial for TRIM22-mediated NF-κB activation. Moreover, our results revealed that TRIM22 overexpression could significantly induce the secretion of pro-inflammatory cytokines by human macrophage cell line U937 in an NF-κB-dependent manner. These data suggested that TRIM22 was a positive regulator of NF-κB-mediated transcription.

T-Cell Receptor Mimic Antibodies for Cancer Immunotherapy.

Antibody-based immunotherapies show clinical effectiveness in various cancer types. However, the target repertoire is limited to surface or soluble antigens, which are a relatively small percentage of the cancer proteome. Most proteins of the human proteome are intracellular. Short peptides from intracellular targets can be presented by MHC class I (MHC-I) molecules on cell surface, making them potential targets for cancer immunotherapy. Antibodies can be developed to target these peptide/MHC complexes, similar to the recognition of such complexes by the T-cell receptor (TCR). These antibodies are referred to as T-cell receptor mimic (TCRm) or TCR-like antibodies. Ongoing preclinical and clinical studies will help us understand their mechanisms of action and selection of target epitopes for immunotherapy. The present review will summarize and discuss the selection of intracellular antigens, production of the peptide/MHC complexes, isolation of TCRm antibodies for therapeutic applications, limitations of TCRm antibodies, and possible ways to advance TCRm antibody-based approaches into the clinic.

Camel nanobodies broadly neutralize SARS-CoV-2 variants.

With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two V H H nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of ≥5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: Dromedary camel ( Camelus dromedarius ) V H H phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.