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Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of “developability.” Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotype-matched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.

BackgroundWith the rapid acceleration of urbanization in China, issues related to urban appearance and public environmental sanitation have become increasingly prominent, and the effectiveness and scientific rigor of relevant policies have attracted widespread attention. Given that China’s governance experience serves as a key reference for developing countries, the quantitative evaluation of these policies holds significant theoretical and practical significance.MethodsThis study aims to conduct a comprehensive analysis, spanning from semantic mining of policy texts in the quantitative assessment, by integrating the Latent Dirichlet Allocation (LDA) and Policy Modeling Consistency (PMC) methods. This approach provides a scientific tool for optimizing urban appearance and public environmental sanitation policies. First, the LDA model was applied to extract themes from policy texts of 159 cities, identifying five core themes: public management tools, governance of citizens’ behaviors, supporting measures, participating subjects, and policy targets. Subsequently, a PMC evaluation system, comprising 9 primary variables and 31 secondary variables, was developed to assign quantitative scores and conduct a visual analysis of policies in 9 representative provinces.ResultsThe results showed that policy quality exhibited a “ladder-like disparity aligned with regional economic levels”: policies in international metropolises and economically vibrant regions mainly were of high quality (with scores ranging from 5.5 to 7.5), while those in less developed areas were generally of low quality (with all scores below 4.5), with a correlation coefficient of 0.82 (p<0.01) between policy quality and regional economic level. Meanwhile, the policy structure was characterized by four imbalances: prioritizing institutional frameworks over implementation details, hardware construction over ideological guidance, long-term planning over short-to-medium-term alignment, and administrative management over public participation.ConclusionThe study indicates that the quality of urban appearance and environmental sanitation policies is significantly correlated with regional economic development, but constrained by structural imbalances. The integrated LDA-PMC approach can effectively support the full-chain analysis of policy texts. In the future, efforts should focus on enhancing inter-regional policy coordination, improving implementation safeguards, refining public participation mechanisms, and establishing a dynamic policy revision system to adapt to the development needs of new-type urbanization.

Automatic computer security inspection of X-ray scanned images has an irresistible trend in modern life. Aiming to address the inconvenience of recognizing small-sized prohibited item objects, and the potential class imbalance within multi-label object classification of X-ray scanned images, this paper proposes a deep feature fusion model-based dual branch network architecture. Firstly, deep feature fusion is a method to fuse features extracted from several model layers. Specifically, it operates these features by upsampling and dimension reduction to match identical sizes, then fuses them by element-wise sum. In addition, this paper introduces focal loss to handle class imbalance. For balancing importance on samples of minority and majority class, it assigns weights to class predictions. Additionally, for distinguishing difficult samples from easy samples, it introduces modulating factor. Dual branch network adopts the two components above and integrates them in final loss calculation through the weighted sum. Experimental results illustrate that the proposed method outperforms baseline and state-of-art by a large margin on various positive/negative ratios of datasets. These demonstrate the competitivity of the proposed method in classification performance and its potential application under actual circumstances.

Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs.

BackgroundThe discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%–30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003).MethodsTo determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further.ResultsPM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo.ConclusionsA unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

In this paper, we conduct an in-depth study and analysis of the suitability of real-time wireless sensor network-assisted smart tourism environment for tourism IoT and evaluate its suitability. We use nodes in wireless sensor networks as relays and collaborative transmission using mutual information accumulation. Using riteless codes, nodes can accumulate information in advance to decode out the original text. Due to the mobility of mobile robots, the planned routing paths can fail quickly, so we propose a dynamic mutual information accumulation collaborative transmission algorithm. For both cases of information transmission from the cloud to the robot and from the robot to the cloud, we dynamically acquire routing paths using neighbour search and chance transmission, respectively. Experimental results show that our dynamic mutual information accumulation collaborative transfer algorithm has lower time complexity and latency. We use a cooperative transmission algorithm with mutual information accumulation, because wireless transmission has the nature of broadcasting, and all neighbour nodes can receive part of the information when the sender starts to transmit information. Riteless codes are used in single-hop transmissions to improve the probability of successful transmissions. Since transmission failure requires waiting for the next neighbour to wake up, which consumes a lot of energy and time in a low duty cycle environment, using riteless codes to improve the transmission success rate of single-hop transmissions can effectively improve the global transmission efficiency. Experimental results show that the opportunity routing algorithm using riteless codes has lower delay and energy consumption. The two major systems, surface, and underground are used to quantitatively measure 23 indicators in five subsystems, including geological environment, soil and water environment, sensitive geological bodies, groundwater resources, and mineral resources, to evaluate the two-way grade of underground space development suitability, revealing the comprehensive bearing characteristics of the study area with an overall high bearing capacity of underground space resources and environment and an overall surplus of bearing status.

The incidence of corpus callosum infarction is low, and sudden cognitive dysfunction caused by corpus callosum infarction is very rare. We report two cases of acute corpus callosum infarction with sudden cognitive impairment, and the related basis, clinical manifestations, diagnosis, differential diagnosis, treatment, and prognosis of this disease are reviewed. The two patients had sudden and severe memory impairment and spatial orientation disorder. Their cognitive function scores were significantly lower, and their MRI demonstrated clear corpus callosum infarction. Through treatment, the symptoms improved significantly. This paper reports two cases with corpus callosum infarction with sudden cognitive impairment, and its relevant background is also reviewed, which will help doctors with the classification diagnosis of cerebral infarction and understanding of corpus callosum infarction.

The development of therapeutic monoclonal antibodies (mAbs) can be hindered by their tendency to aggregate throughout their lifetime, which can illicit immunogenic responses and render mAb manufacturing unfeasible. Consequently, there is a need to identify mAbs with desirable thermodynamic stability, solubility, and lack of self‐association. These behaviors are assessed using an array of in silico and in vitro assays, as no single assay can predict aggregation and developability. We have developed an extensional and shear flow device (EFD), which subjects proteins to defined hydrodynamic forces which mimic those experienced in bioprocessing. Here, we utilize the EFD to explore the aggregation propensity of 33 IgG1 mAbs, whose variable domains are derived from clinical antibodies. Using submilligram quantities of material per replicate, wide‐ranging EFD‐induced aggregation (9‐81% protein in pellet) was observed for these mAbs, highlighting the EFD as a sensitive method to assess aggregation propensity. By comparing the EFD‐induced aggregation data to those obtained previously from 12 other biophysical assays, we show that the EFD provides distinct information compared with current measures of adverse biophysical behavior. Assessing a candidate's liability to hydrodynamic force thus adds novel insight into the rational selection of developable mAbs that complements other assays. The development of therapeutic monoclonal antibodies (mAbs) can be hindered by their tendency to aggregate throughout their lifetime. We have developed an extensional and shear flow device (EFD), which subjects proteins to defined hydrodynamic forces which mimic those experienced in bioprocessing. Here, by comparing the flow‐induced aggregation data to those obtained previously from 12 other biophysical assays, we show that extensional flow provides distinct information compared with current measures of adverse biophysical behavior.

BackgroundCheckpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have paved the way for a new frontier of anti-cancer therapies that modulate our pre-existing immune system to fight against malignancies. 4-1BB is a tumor-necrosis superfamily member expressed on NK and T cells, that acts as a co-stimulatory receptor to improve effector/memory responses towards tumors. Early efforts have focused on the generation of agonist antibodies towards 4-1BB that relied on Fc-mediated cross-linking to cluster and induce receptor downstream signaling, but has led to liver- and immune-related toxicities. We have discovered a PD-L1 x 4-1BB bispecific that exhibits conditional agonist activity in the presence of PD-L1 with better safety features.Methods vHH binders to PD-L1 and 4-1BB were generated from immune libraries derived from camelids and selected via yeast display. Antibody screening was carried out by protein-protein interaction analysis and cell-based binding. Target-related activity was confirmed using luciferase reporter assays. Primary immune cells were also tested for T cell activation via the detection of IL-2 and IFNg secretion. PD-L1-mediated 4-1BB activation via cross-bridging was carried out using target cells expressing PD-L1 co-cultured with effector cells. X-ray crystallography was conducted to resolve the binding sites of both the anti-PD-L1 and anti-4-1BB vHHs. Both tumor efficacy and safety assessment were tested in human knockin mice.ResultsThe 4-1BB binder of PM1003 was found to interact with the CRD4 domain of 4-1BB, as determined by X-ray crystallography. Binding to this domain does not affect the binding between 4-1BB and its ligand 4-1BBL. The anti-PD-L1 vHH binds to an epitope on PD-L1 that overlaps with the binding region of PD-1, and is thus effective in disrupting the interaction between PD-1 and PD-L1. Using luciferase reporter assays and primary cell assays we found the PM1003 could activate 4-1BB in the context of PD-L1-mediated cross-bridging. Data from human 4-1BB and PD-L1 knockin mice also showed that PM1003 could effectively control tumor growth without observing any toxicity signals.ConclusionsPM1003 is a safe and efficacious bispecific antibody that blocks PD-L1 and concurrently activates 4-1BB receptor. An antibody with mild activity was selected directed against the CRD4 domain of 4-1BB to elicit effective potency while minimizing potential toxicity issues. This was reflected in our results. Thus, PM1003 is a potential next generation therapeutic antibody in the IO space that combines and synergizes two independent signaling pathways to control tumor growth.

It has been shown that MDMX inhibits the activity of the tumor suppressor p53 by primarily cooperating with the p53 feedback regulator MDM2. Here, our study shows that this inhibition can be overcome by 14‐3‐3γ and Chk1. 14‐3‐3γ was identified as an MDMX‐associated protein via an immuno‐affinity purification‐coupled mass spectrometry. Consistently, 14‐3‐3γ directly interacted with MDMX in vitro , and this interaction was stimulated by MDMX phosphorylation in vitro and in cells. Interestingly, in response to UV irradiation, the wild‐type, but not the kinase‐dead mutant, Chk1 phosphorylated MDMX at serine 367, enhanced the 14‐3‐3γ‐MDMX binding and the cytoplasmic retaining of MDMX. The Chk1 specific inhibitor UCN‐01 repressed all of these effects. Moreover, overexpression of 14‐3‐3γ, but not its mutant K50E, which did not bind to MDMX, suppressed MDMX‐enhanced p53 ubiquitination, leading to p53 stabilization and activation. Finally, ablation of 14‐3‐3γ by siRNA reduced UV‐induced p53 level and G1 arrest. Thus, these results demonstrate 14‐3‐3γ and Chk1 as two novel regulators of MDMX in response to UV irradiation.