Blocking Activity Research Articles

Abstract 6326: Discovery & preclinical evaluation of BPB-101: A novel triple functional bi-specific antibody targeting GARP-TGFβ complex/SLC, free TGF-β and PD-L1

Abstract Background: Recent studies on tumor microenvironment (TME) have been conferred considerable attention, especially the transforming growth factor-β (TGF-β) signal axis and the PD-L1/PD-1 signal axis. TGF-β is a crucial enforcer in promoting tumor cells angiogenesis, plasticity, fibrosis, and in regulating immune homeostasis and tolerance. The TGF-β signaling controls innate and adaptive immunity in TME by inhibiting the activity of natural killer (NK) cells and effector T cells, while promoting the expansion of regulatory T cells (Tregs). Many studies have revealed that free TGF-β mainly released from glycoprotein A repetitions predominant (GARP)-TGFβ complex at the surface of activated Tregs, B, NK, and tumor cells. Existing antibodies or fusion proteins targeting TGF-β cannot simultaneously block the TGF-β releasing and naturalized free TGF-β in TME, thus limiting the anti-tumor effect. Herein, we designed a bi-specific antibody named as BPB-101 with triple functions: specifically target GARP-TGFβ complex and/or small latent complex (SLC), free TGF-β, and PD-L1. BPB-101 can stop tumor expansion and recruitment, and may enhance the immune effector cells to eliminate the tumor. Methods: An anti-GARP mAb was discovered using a hybridoma platform and selected for binding with GARP-TGFβ complex/SLC and free TGF-β. An anti-PD-L1 nAb was screened out by VHH library and optimized in tumor-bearing mice. After humanizing, these two antibodies were successfully assembled to form a unique bi-specific antibody, BPB-101. The binding affinity of BPB-101 to GARP-TGFβ complex/SLC, free TGF-β and PD-L1 was determined by ELISA, FACS and SPR/BLI. Report gene assay was utilized to detect the blocking activity of BPB-101. The TGF-β secretion from activated Tregs was also analyzed. Finally, the anti-tumor efficacy of BPB-101 was assessed with a transgenic mouse model (B6-hLRRC32). To address the biosafety risks, the nonclinical safety profile was also evaluated in cynomolgus monkeys. Results and Conclusion: BPB-101 is a high-affinity antibody aims to reverse immunosuppressive TME by triple-targeting functions. The blocking ability of TGF-β in not only latent form but also free form makes our BPB-101 distinct from M7824, any other existing anti-TGF-β mAbs and anti-GARP mAbs. In a MC38-hPD-L1 tumor-bearing B6-hLRRC32 model, BPB-101 showed significant tumor growth inhibition of 93 % at the dose of 5 mg/kg (p<0.001), showing superior efficacy to monoclonal antibody alone or in combination with anti-PD-L1 nAb. In addition, BPB-101 showed durable responsiveness and a splendid safety profile with manageable adverse events. These striking results support the clinical development potential of our trifunctional agent as a monotherapy and in combination with other immunotherapies. Citation Format: Wenxin Xu, Jieying Xu, Deyu Xu, Hongjie Cheng, Li Zhang, Huan Zheng, Siyuan Ye, Mengshi Jiang, Jiabing Wang, Lieming Ding. Discovery & preclinical evaluation of BPB-101: A novel triple functional bi-specific antibody targeting GARP-TGFβ complex/SLC, free TGF-β and PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6326.

Abstract 2647: Novel FGFR2 biparatopic antibodies for the treatment of cholangiocarcinoma

Abstract Translocations involving Fibroblast Growth Factor Receptor 2 (FGFR2) are found in up to 45% of Intrahepatic Cholangiocarcinoma (ICC). Treatment of patients bearing FGFR2 fusions with FGFR kinase inhibitors induces tumor regressions, though response rate and durability of response appear limited due to the emergence of resistance or to sub-optimal dosing which is limited by drug adverse effects. Thus, therapeutics that drive activity specifically towards FGFR2 are needed to enhance drug efficacy and reduce adverse effects. We first asked whether the extracellular domain (ECD) of FGFR2-BICC1 fusion is functional and important for receptor dimerization and signaling using NanoBiT system and western blotting. FGFR ECD consists of 3 Ig-fold domains D1, D2, and D3. Using focus transformation and viability assays, we found that deletions in D1, D2, D3, and D2-3 ECD FGFR2-BICC1 decreased growth and transformation of FGFR2 fusion driven cells. Next, we sought to investigate if antibodies against the ECD could perturb the growth of FGFR2 fusion transformed cells. We identified six FGFR2 specific antibodies that bind to epitopes in the ECD and confirmed the ligand blocking activity of these bivalent antibodies. In the absence of ligand, however, most of the bivalent antibodies were relatively inactive. As standard bivalent antibodies engage two receptors at a time, they are at risk for agonism or the maintenance of tonic signaling. Thus, to enhance efficacy and prevent the potential agonist or tonic activity of bivalent antibodies, all possible biparatopic antibodies (15), recognizing two distinct epitopes on the FGFR2 ECD, were engineered from the six parental antibodies. The biparatopic antibodies exhibited enhanced efficacies in FGFR2-fusion driven NIH3T3/BaF3 and ICC13-7 cancer cell lines and significantly improved binding affinities (~10-fold) as analyzed by Octet, MSD-SET, and SPR. Two biparatopic antibodies (BpAb-1, BpAb-2) showed the highest antiproliferative activity and induced apoptosis in FGFR2 fusion driven cells. BpAb-1, BpAb-2 were superior to their parental antibodies in reducing FGFR2-fusion driven growth, transformation and downstream signaling (pFGFR2, pFRS) in vitro and in reducing tumor growth in xenograft models. Importantly, BpAb-1 and BpAb-2 significantly induced FGFR2 internalization and lysosomal-mediated degradation as compared to their parental antibodies. Moreover, BpAb-1 and BpAb-2 had anti-growth activities against ICC patient-derived oncogenic FGFR2 ECD in-frame deletions, and FGFR kinase resistance mutations, including the gatekeeper mutation V565F. BpAb-1 and BpAb-2 were also active in FGFR2 WT amplified cells SNU-16. These results suggest that these FGFR2 biparatopic antibodies are a novel treatment option for ICC patients with tumors harboring FGFR2 fusions. Citation Format: Saireudee Chaturantabut, Sydney Oliver, John Kim, Dennie Frederick, Foxy Robinson, Alessandro Sinopoli, Diego J. Rodriguez, Liang Chang, Nabeel Bardeesy, William Sellers. Novel FGFR2 biparatopic antibodies for the treatment of cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2647.

Abstract 1546: ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models

Abstract Regulatory T (Treg) cells are a distinctive lineage of CD4+ T cells as characterized by expression of FoxP3 and CD25 markers. Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. Accumulation of Treg cells in the tumor microenvironment (TME) is often associated with an adverse prognosis in multiple cancers and also has implication for predicting response to immunotherapy. Treg cell depletion strategy has been explored in preclinical and clinical settings to induce effective antitumor immune responses in TME. CD25, a high-affinity binding subunit alpha of the IL-2 receptor, is constitutively and highly expressed on Treg cells but also transiently upregulated on effector T cells. The high level of CD25 expression on Treg cells could deprive of IL-2 for effector T cells and inhibit their proliferation. Thus, an effective approach to selectively suppress Treg cell function is to deplete Treg cells while avoid blocking IL-2 binding to CD25 for allowing T effect cell proliferation and activation. Using our immune tolerance breaking antibody platform, we generated a humanized IgG1 subtype anti-CD25 monoclonal antibody (TST010). TST010 bound to human CD25 with high affinity and selectivity but did not block IL-2 binding to CD25. No blocking activity to IL-2 signaling on human PBMC was further confirmed by the phosphorylation of Stat5 using FACS analysis. By reducing fucosylation during cell culture process, TST010 mAb gained the enhanced ADCC activity. In an ADCC reporter assay, TST010 showed its sub-nanomolar ADCC activity against CD25-overexpression SU-DHL-1 cell line and human induced Treg cells, but much less to human activated CD8+ T effect cells due to lower CD25 expression. Similarly, in a NK cytotoxicity assay, TST010 selectively killed human induced Treg cells in contrast to activated CD8+ T cells. In vivo antitumor activity of TST010 was evaluated in the syngeneic mouse tumor models. The mouse colon cancer MC38 model was developed on the human CD25 knock-in mice. In the MC38 tumor model, TST010 markedly reduced the Treg (FoxP3+CD4+) population in both peripheral blood and TME. Importantly, the CD8+T/Treg ratio in tumor was increased starting at 2 weeks post TST010 treatment, and it resulted in a significant single agent tumor growth inhibition at a 10 mg/kg dose. Furthermore, in the LLC tumor model which does not respond well to checkpoint inhibitors, the anti-CD25 mAb showed a good combination effect with a PD-L1 inhibitor. In summary, we have discovered a novel therapeutic anti-CD25 mAb that can deplete Treg cells without blocking IL-2 signaling. As demonstrated in our preclinical tumor models, it has a good potential to induce effective antitumor immune responses in TME and tumor growth inhibition especially in combination with PD-1/PD-L1 treatment. Citation Format: Di Sun, Huanhuan Guo, Hongjun Li, Xinlai Yao, Yiqing Xu, Shenjie Zhang, Shijie Zhou, Xiaoli Zi, Lisa Zheng, Yi Gu, Xueming Qian. ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1546.

LNA blockers for improved amplification selectivity

LNA-containing oligonucleotides bind DNA more tightly than standard DNA, so they can interact with targeted sequences and affect multiple processes. When a desired DNA is present at low concentrations relative to nearly identical undesired DNAs, LNAs can block amplification of unwanted DNAs. Using a short rAAV and synthetic DNA sequence as a model, we studied the length, number, and positioning of LNA bases to improve blocker effectiveness. Oligonucleotides 18–24 bases long with LNAs at every other position were most effective. Highly degenerate targets were used to characterize the impact of mismatches on blocking. Mismatches at LNA ends had little impact on blocking activity. Single and double mismatches were tolerated with longer blockers, especially if the mismatches were near LNA ends. Shorter LNAs were more selective, with > 1 mismatch preventing effective blocking. Neither the strand to which a blocker bound nor the distance between the blocker and priming sites greatly impacted blocking efficiency. We used these findings to design blockers of wild-type DNA versus the single-base A1AT PiZ allele. Blockers are most specific when the mismatch is located away from the LNA 5′ end. Pairs of partially overlapping blockers on opposite strands with a centrally-located mismatch have maximal activity and specificity.

High Dominant Frequencies and Fractionated Potentials Do Not Indicate Focal or Rotational Activation During AF

BackgroundDominant frequencies (DFs) or complex fractionated atrial electrograms (CFAEs), indicative of focal sources or rotational activation, are used to identify target sites for atrial fibrillation (AF) ablation in clinical studies, although the relationship among DF, CFAE, and activation patterns remains unclear. ObjectivesThis study sought to investigate the relationship between patterns of activation underlying DF and CFAE sites during AF. MethodsEpicardial high-resolution mapping of the right and left atrium including Bachmann’s bundle was performed in 71 participants. We identified the highest dominant frequency (DFmax) and highest degree of CFAE (CFAEmax) with the use of existing clinical criteria and classified patterns of activation as focal or rotational activation and smooth propagation, conduction block (CB), collision and remnant activity, and fibrillation potentials as single, double, or fractionated potentials containing, respectively, 1, 2, or 3 or more negative deflections. Relationships among activation patterns, DFmax, and potential types were investigated. ResultsDFmax were primarily located at the left atrioventricular groove and did not harbor focal activation (proportion focal waves: 0% [IQR: 0%-2%]). Compared with non-DFmax sites, DFmax were characterized by more frequent smooth propagation (22% [IQR: 7%-48%] vs 17% [IQR: 11%-24%]; P = 0.001), less frequent conduction block (69% [IQR: 51%-81%] vs 74% [IQR: 69%-78%]; P = 0.006), a higher proportion of single potentials (72% [IQR: 55%-84%] vs 6%1 [IQR: 55%-65%]; P = 0.003), and a lower proportion of fractionated potentials (4% [IQR: 1%-11%] vs 12% [IQR: 9%-15%]; P = 0.004). CFAEmax were mainly found at the pulmonary veins area, and only 1% [IQR: 0%-2%] of all CFAEmax contained focal activation. Compared with non-CFAEmax sites, CFAEmax sites were characterized by less frequent smooth propagation (1% [IQR: 0%-1%] vs 17% [IQR: 12%-24%]; P < 0.001) and more frequent remnant activity (20% [IQR: 12%-29%] vs 8% [IQR: 5%-10%]; P < 0.001), and harbored predominantly fractionated potentials (52% [IQR: 43%-66%] vs 12% [IQR: 9%-14%]; P < 0.001). ConclusionsFocal or rotational patterns of activation were not consistently detected at DFmax domains and CFAEmax sites. These findings do not support the concept of targeting DFmax or CFAEmax according to existing criteria for AF ablation.

Chromatography analysis, in light of vitro antioxidant, antidiabetic, antiobesity, anti-inflammatory, antimicrobial, anticancer, and three-dimensional cancer spheroids’ formation blocking activities of Laurus nobilis aromatic oil from Palestine

Laurus nobilis (LN) has been used throughout the years as a food flavoring and in traditional medicine. The LN leaves have various biological activities, such as antioxidant, wound healing, antibacterial, analgesic, and anti-inflammatory activities. However, oxidative stress, cancer, diabetes, microbial infections, and inflammatory diseases are closely linked. The objective of this research is to characterize Laurus nobilis (LN) aromatic oil (AO) and evaluate its antioxidant, antidiabetic, antiobesity, antimicrobial, and antimutagenic bioactivities. The AO constituents were characterized using gas chromatography–mass spectrometry (GC–MS). The antimicrobial activity was performed using a microdilution assay against six common microbial species. Free radicals, a porcine pancreatic lipase, α-amylase, and α-glucosidase inhibitory assays were conducted utilizing reference biomedical methods. The cytotoxic effect of LNAO was established on a variety of cancer and normal cell lines using the MTS assay. The anti-inflammatory activity of LNAO was evaluated using the Cayman COX activity kit. The results indicate about 99% of the total oil is composed of 36 compounds, the characterized AO metabolites showed content of many oxygenated terpenoids with 1,8-Cineole and Terpinyl acetate as a major component with a percentage of (40.39 and 15.07, respectively. The plant AO showed potent antioxidant activity (IC50 = 2.2 ± 1.38) and has moderate anti-amylase (IC50 = 60.25 ± 1.25), anti-glucosidase (IC50 = 131.82 ± 0.1), and antilipase (IC50 = 83.17 ± 0.06) activities. Moreover, LNAO showed potent antimicrobial activity against Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Proteus vulgaris (MICs = 1.56 µg/mL), methicillin-resistant Staphylococcus aureus (MRSA) (MIC = 3.125 µg/mL) and Candida albicans (MIC = 0.195 µg/mL). The cytotoxicity results demonstrated that at a concentration of 1 mg/mL, LNAO has potent breast cancer (MCF-7), and hepatocellular carcinoma (Hep 3B) cancer cells inhibitory activities of 98% and 95%, respectively. Importantly, we are the first to show that LNAO significantly hinders hepatocellular carcinoma spheroids’ formation capacity in a 3D model. These results show that LNAO is a promising natural source with powerful antioxidant, antidiabetic, anticancer, and antimicrobial activities that could be exploited in the future to treat a variety of diseases.Graphical

Impact of anomalous Eurasian blocking activities on the East Asian Meiyu rainfall

An extreme East Asian Meiyu rainfall in both amount and duration occurred along Yangtze River valley during June–July of 2020, however, possible mid-high latitude signatures causing this super Meiyu have not been well identified. This study explores the cause of the Meiyu rainfall from the aspect of anomalous Eurasian blocking activities with a two-dimensional blocking index, using the Japanese 55-yr Reanalysis for 1979–2020. The major findings are as follows. Variabilities of the Eurasian blocking activities are primarily characterized by a tripole pattern with three centers over the Baltic Sea, the Ural Mountains, and the Sea of Okhotsk, respectively. The tripole pattern is associated with two zonally-oriented Rossby wavetrains which may originate from the rainfall anomaly in central Europe. Corresponding to a positive phase of the tripole pattern, the northern wavetrain through energy dispersion tends to induce an anomalous anticyclone (i.e., enhanced blocking) over the Sea of Okhotsk, while the southern wavetrain tends to induce an anomalous cyclone over the Sea of Japan which is conducive to a southward intrusion of more high-latitude cold airs. Consequently, a meridional dipole anomalous circulation pattern over the northeastern Asia is formed, favoring more East Asian Meiyu rainfall. In 2020, the Eurasian blocking activities exhibit a significantly positive phase of the tripole pattern and considerably contribute to the super Meiyu. The results identified in this study highlight the importance of the Eurasian blocking activities in the East Asian Meiyu and provide a new perspective for the prediction of the Meiyu rainfall with mid-high latitude signatures.

Intense Wildfires in Russia over a 22-Year Period According to Satellite Data

The spatiotemporal distributions of wildfire areas and FRP values for the territory of Russia and its large regions (the European part of Russia, as well as the Ural, Siberian, and Far Eastern Federal Districts) during 2001–2022 were analyzed using satellite data. For the territory of Russia, there was a decreasing trend in annual burned areas and a small increase in average hotspot FRP. At the same time, the largest annual burned areas in the territory of Russia were recorded in 2008 (295.2 thous. km2), 2002 (272.4 thous. km2), 2006 (261.2 thous. km2), and in 2012 (258.4 thous. km2). It was found that during the studied period, 90% of fire hotspots in Russia had a maximum FRP &lt; 100 MW. The most intense wildfires (FRP &gt; 1500 MW) amounted to only 0.1% and were detected mainly in the Siberian and Far Eastern Federal Districts. Interconnections between large wildfires and meteorological factors, including blocking activity in the atmosphere, were revealed.

Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.

Keratitis ichthyosis deafness (KID) syndrome is a rare disorder caused by hemichannel (HC) activating gain-of-function mutations in the GJB2 gene encoding connexin (Cx) 26, for which there is no cure, or current treatments based upon the mechanism of disease causation. We applied Adeno Associated Virus (AAV) mediated mAb gene transfer (AAVmAb) to treat the epidermal features of KID syndrome with a well-characterized HC blocking antibody using male mice of a murine model that replicates the skin pathology of the human disease. We demonstrate that invivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis invivo. We also show that AAVmAb treatment eliminated abnormal keratinocyte proliferation and enlarged cell size, decreased apoptosis, and restored the normal distribution of keratin expression. Our findings reinforce the critical role played by increased HC activity in the skin pathology associated with KID syndrome. They also underscore the clinical potential of anti-HC mAbs coupled with genetic based delivery systems for treating the underlying mechanistic basis of this disorder. Inhibition of HC activity is an ideal therapeutic target in KID syndrome, and the genetic delivery of mAbs targeted against mutant HCs could form the basis of new therapeutic interventions to treat this incurable disease. Fondazione Telethon grant GGP19148 and University of Padova grant Prot. BIRD187130 to FM; Foundation for Ichthyosis and Related Skin Types (FIRST) and National Institutes of Health grant EY 026911 to TWW.

Fibroblast, Epithelial and Endothelial Cell-Derived Human Cytomegalovirus Strains Display Distinct Neutralizing Antibody Responses and Varying Levels of gH/gL Complexes.

In sequential sera from pregnant women with HCMV primary infection (PI), the serum neutralizing activity is higher against virions produced in epithelial and endothelial cells than in fibroblasts. Immunoblotting shows that the pentamer complex/trimer complex (PC/TC) ratio varies according to the producer cell culture type used for the virus preparation to be employed in the neutralizing antibody (NAb) assay, and is lower in fibroblasts and higher in epithelial, and especially endothelial cells. The blocking activity of TC- and PC-specific inhibitors varies according to the PC/TC ratio of virus preparations. The rapid reversion of the virus phenotype following its back passage to the original cell culture (fibroblasts) potentially argues in favor of a producer cell effect on virus phenotype. However, the role of genetic factors cannot be overlooked. In addition to the producer cell type, the PC/TC ratio may differ in single HCMV strains. In conclusion, the NAb activity not only varies with different HCMV strains, but is a dynamic parameter changing according to virus strain, type of target and producer cells, and number of cell culture passages. These findings may have some important implications for the development of both therapeutic antibodies and subunit vaccines.

Identification of CBPA as a New Inhibitor of PD-1/PD-L1 Interaction.

Targeting of the PD-1/PD-L1 immunologic checkpoint is believed to have provided a real breakthrough in the field of cancer therapy in recent years. Due to the intrinsic limitations of antibodies, the discovery of small-molecule inhibitors blocking PD-1/PD-L1 interaction has gradually opened valuable new avenues in the past decades. In an effort to discover new PD-L1 small molecular inhibitors, we carried out a structure-based virtual screening strategy to rapidly identify the candidate compounds. Ultimately, CBPA was identified as a PD-L1 inhibitor with a KD value at the micromolar level. It exhibited effective PD-1/PD-L1 blocking activity and T-cell-reinvigoration potency in cell-based assays. CBPA could dose-dependently elevate secretion levels of IFN-γ and TNF-α in primary CD4+ T cells in vitro. Notably, CBPA exhibited significant in vivo antitumor efficacy in two different mouse tumor models (a MC38 colon adenocarcinoma model and a melanoma B16F10 tumor model) without the induction of observable liver or renal toxicity. Moreover, analyses of the CBPA-treated mice further showed remarkably increased levels of tumor-infiltrating CD4+ and CD8+ T cells and cytokine secretion in the tumor microenvironment. A molecular docking study suggested that CBPA embedded relatively well into the hydrophobic cleft formed by dimeric PD-L1, occluding the PD-1 interaction surface of PD-L1. This study suggests that CBPA could work as a hit compound for the further design of potent inhibitors targeting the PD-1/PD-L1 pathway in cancer immunotherapy.

Autoantibodies are highly prevalent in non-SARS-CoV-2 respiratory infections and critical illness.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

Trimming the disordered C-terminal extension of the protease inhibitor Hg1 produces a potent Kv1.3 blocker.

Animal venom contains a mixture of toxic polypeptides that act in synergy to enable the fast immobilization of prey or deter predators and competitors. This polypeptide arsenal is constantly replenished by recruiting protein scaffolds from various organs into the venom gland. The recruited proteins often retain their ancestral functions but acquire specific modifications associated with novel toxic functions. Here we report an in-depth analysis of Hg1, a Kunitz-fold protease inhibitor with moderate blocking activity on Kv1.3, isolated from the venom gland of the Hadrurus gertschi scorpion. Electrophysiological analyses, supported by MD simulations, suggested that in contrast to canonical K+ blocking peptides, Hg1 does not insert a lysine residue into the channel pore. The crystal structure of the toxin revealed an unusually long, disordered extension at the C-terminal region of the molecule. Deletion of the C-terminal extension increased the potency of the toxin by order of magnitude and restored the voltage sensitivity of the block, a hallmark of the canonical block mechanism. In silico analysis supported the notion that removal of the disordered C-terminal extension has lifted a geometric restraint that prevents the wild-type toxin from effectively blocking Kv1.3. We propose that Hg1 is an evolutionary intermediate between a potent protease inhibitor and a potent K+ channel blocker.

A pH-dependent anti-CD47 antibody that selectively targets solid tumors and improves therapeutic efficacy and safety

BackgroundThe antiphagocytic molecule CD47 is overexpressed in a wide variety of cancer cells, and antibodies targeting CD47 for cancer therapies are currently under intensive investigation. However, owing to the ubiquitous expression of CD47 on healthy cells, anti-CD47 therapies often achieve only weak therapeutic benefits and can induce severe side effects. Here, we report the generation of a pH-dependent anti-CD47 antibody (BC31M4) which selectively binds to tumors under the acidic solid tumor microenvironment.MethodsBC31M4 was generated using antibody phage display and a pH-dependent selection strategy. The pH-dependent binding and blocking activities of BC31M4 were verified using in vitro assays, and the structural basis of the pH-dependent binding property was characterized. BC31M4’s antitumor effect was confirmed by both phagocytosis assays and studies in xenograft models. The tumor selectivity, mechanism of action, PK properties, side effects, and therapeutic efficacy were further evaluated in humanized (hCD47 and its receptor hSIRPα) immunocompetent syngeneic mouse models.ResultsThe crystal structure reveals that two histidines locate within the CDRs of the light chain directly contribute to the pH-dependent binding of BC31M4. BC31M4 promotes macrophage phagocytosis of tumor cells more potently at acidic-pH than at physiological-pH. Our hCD47/hSIRPα humanized syngeneic mouse model results demonstrated that BC31M4 selectively accumulates in tumors but not in normal tissues. BC31M4 causes minimal side effects and exhibits superior PK properties as compared to the other examined anti-CD47 antibodies. When combined with adoptive T cell transfer, BC31M4 efficiently promotes adaptive immune responses against tumors and also induces immune memory. Moreover, we show that BC31M4’s antitumor effects rely on an Fc that mediates strong effector functions.ConclusionsOur study illustrates that the development of a tumor-selective, pH-dependent anti-CD47 antibody safely confers strong therapeutic effects against solid tumors, thus providing a promising therapeutic strategy to overcome the challenges of anti-CD47 therapy.

The role of IgG1 and IgG4 as dominant IgE-blocking antibodies shifts during allergen immunotherapy

The induction of allergen-specific IgE-blocking antibodies is a hallmark of allergen immunotherapy (AIT). The inhibitory bioactivity has largely been attributed to IgG4; however, our recent studies indicated the dominance of IgG1 early in AIT. Here, the IgE-blocking activity and avidity of allergen-specific IgG1 and IgG4 antibodies were monitored throughout 3 years of treatment. Serum samples from 24 patients were collected before and regularly during AIT with birch pollen. Bet v 1-specific IgG1 and IgG4 levels were determined by ELISA and ImmunoCAP, respectively. Unmodified and IgG1- or IgG4-depleted samples were compared for their inhibition of Bet v 1-induced basophil activation. The stability of Bet v 1-antibody complexes was compared by ELISA and by surface plasmon resonance. Bet v 1-specific IgG1 and IgG4 levels peaked at 12 and 24 months of AIT, respectively. Serological IgE-blocking peaked at 6 months and remained high thereafter. In the first year of therapy, depletion of IgG1 clearly diminished the inhibition of basophil activation while the absence of IgG4 hardly reduced IgE-blocking. Then, IgG4 became the main inhibitory isotype in most individuals. Both isotypes displayed high avidity to Bet v 1 ab initio of AIT, which did not increase during treatment. Bet v 1-IgG1 complexes were enduringly more stable than Bet v 1-IgG4 complexes were. In spite of the constant avidity of AIT-induced allergen-specific IgG1 and IgG4 antibodies, their dominance in IgE-blocking shifted in the course of treatment. The blocking activity of allergen-specific IgG1 should not be underestimated, particularly early in AIT.

Anti-ageing effects of FDA-approved medicines: a focused review.

Ageing is the process generated by senescent cells, free radicals, inflammation and other relevant factors. Ageing contributes to age-related diseases that affect the quality of life. People are interested in anti-ageing intervention and many scientists attempt to search for anti-ageing medicines. This review focused on describing invivo anti-ageing activity of US-FDA-approved drugs and found that alogliptin, canagliflozin and metformin might produce anti-ageing activity via AMPK activation. Rapamycin and canagliflozin are capable to inhibit mTOR to promote lifespan. Atracurium, carnitine and statins act as DAF-16 activators, which potentially contribute to anti-ageing activity. Hydralazine, lisinopril, rosiglitazone and zidovudine may help stabilize genomic integrity to prolong life expectancy. Other indirect mechanisms, including insulin-lowering effect by acarbose and calcium channel blocking activity by verapamil may also promote longevity. Interestingly, some drugs (i.e.,canagliflozin, metformin, rapamycin and acarbose) are likely to demonstrate a lifespan-promoting effect predominantly in male animals. These pre-clinical data might provide mechanistic and phenotypic perspectives to better understand the targets of anti-ageing interventions.

Myocardial work and energy loss of left ventricle obtained by pressure-strain loop and vector flow mapping: a new perspective on idiopathic left bundle branch block

To date, no research has been conducted on the electrical activity and mechanical dyssynchrony of idiopathic left bundle branch block (iLBBB) with normal left ventricular ejection fraction (LVEF). This study sought to assess the left ventricular summation of energy loss (EL-SUM) and average energy loss (EL-AVE) using vector flow mapping as well as myocardial work using pressure-strain loop (PSL) in patients with iLBBB and normal LVEF. We prospectively recruited 35 patients with iLBBB and 35 control participants with normal LVEF. Echocardiography was performed. Conventional echocardiographic parameters, myocardial work, and energy loss (i.e., the EL-SUM and EL-AVE) were calculated. In relation to global myocardial work, compared to the control participants, the iLBBB patients showed decreased global longitudinal strain (GLS; -15.32%±2.58% vs. -18.27%±2.12%; P=0.001), a decreased global work index (GWI; 1,428.24±338.18 vs. 1,964.87±264.16 mmHg%; P<0.001), decreased global work efficiency (GWE) (84.48±5.19 vs. 91.73±5.31 mmHg%; P<0.001), and significantly increased global waste work (GWW; 341.60±132.62 vs. 161.80±106.81 mmHg%; P<0.001). In relation to the regional index, the iLBBB patients had a significantly reduced basal anteroseptal segment (879.15±370.50 vs. 1,746.38±154.44 mmHg%; P<0.001), basal inferoseptal segment (1,111.42±389.04 vs. 1,677.25±223.10 mmHg%; P<0.001), mid-anteroseptal segment (1,097.54±394.83 vs. 1,815.06±291.22 mmHg%; P<0.001), mid-inferoseptal segment (1,012.54±353.33 vs. 1,880.88±254.39 mmHg%; P<0.001), apical anterior segment (1,592.42±366.64 vs. 1,910.00±170.27 mmHg%; P=0.001), apical lateral segment (1,481.62±342.95 vs. 1,817.19±227.55 mmHg%; P=0.001), apical septal segment (1,437.65±428.22 vs. 1,852.25±275.19 mmHg%; P=0.001), and apex (1,542.62±342.89 vs. 1,907.06±197.94 mmHg%; P<0.001). The iLBBB patients had increased EL-AVE and EL-SUM during the late-diastole, isovolumic-systole, and rapid-ejection periods [EL-AVE in T2: 28.3 (8.7, 49.0) vs. 6.8 (5.4, 9.4) J/(s·m3); P=0.029]; [EL-AVE in T3: 24.7 (13.0, 46.8) vs. 7.2 (5.4, 10.8) J/(s·m3), P<0.001]; [EL-AVE in T4: 18.3 (12.0, 27.6) vs. 7.7 (4.1, 11.6) J/(s·m3), P=0.002]; [EL-SUM in T2: 8.3 (2.2, 14.5) vs. 2.1 (1.6, 3.2) J/(s·m), P=0.049]; [EL-SUM in T3: 7.6 (4.0, 14.5) vs. 2.2 (1.7, 3.3) J/(s·m), P<0.001]; [EL-SUM in T4: 5.3 (3.6, 9.7) vs. 2.2 (1.4, 3.0) J/(s·m), P=0.004]. The GWI and GWE were reduced in patients with iLBBB, especially in the septum and apex. The EL-SUM and EL-AVE were higher in patients with iLBBB during the late-diastole, isovolumic-systole, and rapid-ejection periods. EL and PSL reflect the LV hemodynamics of patients with iLBBB.

Membrane-disruptive homo-polymethacrylate with both hydrophobicity and pH-sensitive protonation for selective cancer therapy.

The membrane-disruptive strategy, which involves host defense peptides and their mimetics, is a revolutionary cancer treatment based on broad-spectrum anticancer activities. However, clinical application is limited by low selectivity towards tumors. In this context, we have established a highly selective anticancer polymer, i.e. poly(ethylene glycol)-poly(2-azepane ethyl methacrylate) (PEG-PAEMA), that can mediate the membrane-disruptive activity via a subtle pH change between physiological pH and tumor acidity for selective cancer treatment. Specifically, the resulting PEG-PAEMA can assemble into neutral nanoparticles and silence the membrane-disruptive activity at physiological pH and disassemble into cationic free-chains or smaller nanoparticles with potent membrane-disruptive activity after the protonation of the PAEMA block due to tumor acidity, resulting in high selectivity towards tumors. Dramatically, PEG-PAEMA exhibited a >200-fold amplification in hemolysis and <5% in IC50 against Hepa1-6, SKOV3 and CT-26 cells at pH 6.7 as compared to those at pH 7.4, thanks to the selective membrane-disruptive mechanism. Moreover, mid- and high-dose PEG-PAEMA demonstrated higher anticancer efficacy than an optimal clinical prescription (bevacizumab plus PD-1) and, significantly, had few side effects on major organs in the tumor-bearing mice model, agreeing with the highly selective membrane-disruptive activity in vivo. Collectively, this work showcases the latent anticancer pharmacological activity of the PAEMA block, and also brings new hope for selective cancer therapy.

An Efficient Low Complexity Region-of-Interest Detection for Video Coding in Wireless Visual Surveillance

Following its use in several applications, including video coding in wireless surveillance, moving object detection (MOD) has become a popular video analysis topic. Despite the considerable progress in the accuracy of MOD for video coding, its implementation in constrained sensors is a real challenge owing to their high complexity and energy consumption. Therefore, there is a great need to address the trade-off between the accuracy and the energy efficiency of MOD approaches for video coding in constrained systems. In this work, an energy-efficient region-of-interest (ROI) detection algorithm as a pre-encoder for wireless visual surveillance (WVS) is proposed. The algorithm ensures a trade-off between detection accuracy and computational complexity. To this end, we propose constructing an activity map by measuring each block activity between successive frames. The map scores are processed using a combination of a fast Gaussian smoother and a rank-order filter to improve accuracy. Only the blocks in motion are coded and counted for transmission. The accuracy of our approach has been evaluated on a large dataset using key performance metrics. It has been found that our algorithm outperforms other state-of-the-art techniques in terms of true positive rate (TPR), with 80.84% on sensitivity metric, while exhibiting a well-balanced accuracy for all categories. A careful examination of the computational complexity confirms the low overhead. The energy and bitrate savings could achieve nearly 90% and 98%, respectively.

New supramolecules of bis(acylhydrazones)-linked bisphenol sulfide for Alzheimer's: targeting cholinesterases by in vitro and in silico approaches.

In current research, two functional components, i.e., hydrazone and bisphenol sulfide were combined to get useful supramolecules in medicinal chemistry. Herein 25 new 4,4'-thiodiphenol bis-acylhydrazones were synthesized in good to excellent yields. Initially ethyl-2-chloroacetate was reacted with 4,4'-thiodiphenol, which was further reacted with excess hydrazine hydrate to produce 2,2'-((thiobis(4,1-phenylene))bis(oxy))di(acetohydrazide), which was then combined with various aromatic and aliphatic aldehydes to get the desired products (hydrazones, 4a-4y). The synthesized supramolecules were characterized by contemporary spectroscopic techniques such as 1H NMR, 13C NMR, and mass spectroscopy. The synthetic compound's cholinesterase blocking activity was tested against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes where compounds 4n, and 4h showed excellent inhibitory potential for AChE, while 4b, and 4h, demonstrated most potent inhibition of BChE. The starting compound (SM3) and compounds 4h and SM3 depicted excellent dual inhibitory capabilities for both enzymes. The chemical basis of anticholinesterase activity was investigated using a structure-based molecular docking approach. The biological significance and the ease of synthesis of this class of compounds should be considered in therapeutic development for Alzheimer's disease treatments.