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Background: B7-H3, a type I transmembrane glycoprotein belonging to the B7 superfamily, is an attractive target for antitumor therapies. B7-H3 demonstrates aberrant overexpression in various types of solid tumors while showing limited and low expression in normal human organs. Various types of treatment targeting B7-H3 have been reported. Among these treatments, antibody–drug conjugates (ADCs) have shown potent activity, and several clinical trials, including DS7300a and MGC018, are currently ongoing. Methods: Here, we constructed CD276-8 ADC, composed of the anti-B7-H3 antibody CD276-8 with moderate affinity, an enzymatically cleavable tetra-peptide-based linker and DXd. Characteristics, including in vitro binding affinity and internalization activity, were assessed by bio-layer interferometry (BLI), flow cytometry and high content analysis (HCA). The cytotoxicity of CD276-8 ADC was evaluated in cell lines expressing B7-H3. Pharmacokinetic profiles and antitumor activity were evaluated in mouse models in vivo. Finally, the developability of CD276-8 ADC was assessed with plasma stability, accelerated stability and freeze–thaw studies using LC-MS and HPLC. Results: Characterization in vitro demonstrated the moderate affinity and acceptable internalization activity of CD276-8 ADC. In addition, CD276-8 ADC exhibited potent antitumor activities in B7-H3-positive cell line-derived xenograft (CDX) models with acceptable pharmacokinetic profiles, although it showed less potent cytotoxicity in various cell lines in vitro, indicating acceptable developability. Conclusions: We developed CD276-8 ADC, a B7-H3-targeting ADC with moderate affinity, which delivers the TOP1 inhibitor DXd. This design combined moderate affinity and acceptable pharmacokinetics, resulting in potent antitumor efficacy in vivo. Our study suggests that affinity optimization could be a useful consideration for enhancing ADC efficacy, positioning CD276-8 ADC as a promising therapeutic for B7-H3-expressing solid tumors.

Background: Antibodies and derivative drugs targeting immune checkpoints have been approved for the treatment of several malignancies, but there are fewer responses in patients with pancreatic cancer. Here, we designed a nanobody molecule with bi-targeting on PD-L1 and CXCR4, as both targets are overexpressed in many cancer cells and play important roles in tumorigenesis. We characterized the biochemical and anti-tumour activities of the bispecific nanobodies in vitro and in vivo. Methods: A nanobody molecule was designed and constructed. The nanobody sequences targeting PD-L1 and CXCR4 were linked by the (G 4 S) 3 flexible peptide to construct the anti-PD-L1/CXCR4 bispecific nanobody. The bispecific nanobody was expressed in E. coli cells and purified by affinity chromatography. The purified nanobody was biochemically characterized by mass spectrometry, Western blotting and flow cytometry to confirm the molecule and its association with both PD-L1 and CXCR4. The biological function of the nanobody and its anti-tumour effects were examined by an in vitro tumour cell-killing assay and in vivo tumour inhibition in mouse xenograft models. Results: A novel anti-PD-L1/CXCR4 bispecific nanobody was designed, constructed and characterized. The molecule specifically bound to two targets on the surface of human cancer cells and inhibited CXCL12-induced Jurkat cell migration. The bispecific nanobody increased the level of IFN-γ secreted by T-cell activation. The cytotoxicity of human peripheral blood mononuclear cells (hPBMCs) against pancreatic cancer cells was enhanced by the molecule in combination with IL-2. In a human pancreatic cancer xenograft model, the anti-PD-L1/CXCR4 nanobody markedly inhibited tumour growth and was superior to the combo-treatment by anti-PD-L1 nanobody and anti-CXCR4 nanobody or treatment with atezolizumab as a positive control. Immunofluorescence and immunohistochemical staining of xenograft tumours showed that the anti-tumour effects were associated with the inhibition of angiogenesis and the infiltration of immune cells. Conclusion: These results clearly revealed that the anti-PD-L1/CXCR4 bispecific nanobody exerted anti-tumour efficacy in vitro and inhibited tumour growth in vivo. This agent can be further developed as a therapeutic reagent to treat human pancreatic cancer by simultaneously blocking two critical targets.

Many studies have been conducted on the prediction of nitrous oxide (N2O) emissions from soils. Comparably, prediction of N2O water–air emissions is much more limited, especially at the national level. Here, we collected published N2O emission data across China's watersheds and analyzed spatiotemporal patterns during dry and wet seasons. We predicted N2O emission fluxes from these waterbodies for 2026–2028 using a traditional gray prediction model (GM) coupled with several deep learning models: Long Short‐Term Memory (LSTM), Gated Recurrent Unit (GRU), and Bidirectional Long Short‐Term Memory (BiLSTM). The study showed large regional variation in emissions from subtropical to boreal watersheds. Average emission rates varied from 13.95 (±27.15) μg m−2 h−1 in the Yellow River Basin to 68.71 (±102.62) μg m−2 h−1 in Southwest China. N2O emissions were clearly higher in the dry season than the wet season in all regions except the Yellow River Basin, indicating strong influence from wetland vegetation. Regarding model performance, higher accuracy was achieved by GRU and BiLSTM, which successfully predicted fluctuating increases of N2O emission fluxes in most regions from 2026 to 2028, reflecting seasonal changes. While LSTM performed less accurately, GRU and BiLSTM, evolved from LSTM, may be more appropriate for complex situations. These findings provide insights into national spatiotemporal patterns of N2O emissions and can guide regional and national mitigation strategies as well as future research.

Background Though tamoxifen achieves success in treating estrogen receptor α (ERα)-positive breast cancer, the followed development of tamoxifen resistance is a common challenge in clinic. Signals downstream of prolactin receptor (PRLR) could synergize with ERα in breast cancer progression. However, the potential effect of targeting PRL-PRLR axis combined with tamoxifen has not been thoroughly investigated. Methods High-throughput RNA-seq data obtained from TCGA, Metabric and GEO datasets were analyzed to explore PRLR expression in breast cancer cell and the association of PRLR expression with tamoxifen treatment. Exogenous or PRL overexpression cell models were employed to investigate the role of activated PRLR pathway in mediating tamoxifen insensitivity. Immunotoxin targeting PRLR (N8-PE24) was constructed with splicing-intein technique, and the efficacy of N8-PE24 against breast cancer was evaluated using in vitro and in vivo methods, including analysis of cells growth or apoptosis, 3D spheroids culture, and animal xenografts. Results PRLR pathway activated by PRL could significantly decrease sensitivity of ERα-positive breast cancer cells to tamoxifen. Tamoxifen treatment upregulated transcription of PRLR and could induce significant accumulation of PRLR protein in breast cancer cells by alkalizing lysosomes. Meanwhile, tamoxifen-resistant MCF7 achieved by long-term tamoxifen pressure exhibited both upregulated transcription and protein level of PRLR. Immunotoxin N8-PE24 enhanced sensitivity of breast cancer cells to tamoxifen both in vitro and in vivo. In xenograft models, N8-PE24 significantly enhanced the efficacy of tamoxifen and paclitaxel when treating PRLR-positive triple-negative breast cancer. Conclusions PRL-PRLR axis potentially associates with tamoxifen insensitivity in ERα-positive breast cancer cells. N8-PE24 could inhibit cell growth of the breast cancers and promote drug sensitivity of PRLR-positive breast cancer cells to tamoxifen and paclitaxel. Our study provides a new perspective for targeting PRLR to treat breast cancer. Highlights Tamoxifen up-regulates PRLR protein level in breast cancer cells and activation of PRLR pathway by PRL could decrease drug-sensitivity of breast cancer cells to tamoxifen. The immunotoxin targeting PRLR could reverse drug-sensitivity to tamoxifen in tamoxifen-resistant breast cancer in vitro and in vivo. The immunotoxin targeting PRLR significantly improve the efficacy of chemotherapy in PRLR-positive TNBC and xenograft models.

Many methods have been developed to produce bispecific antibodies (BsAbs) for industrial application. However, huge challenges still remain in synthesizing whole length BsAbs, including their assembly, stability, immunogenicity, and pharmacodynamics. Here we present for first time a generic technology platform of generating bispecific IgG antibodies, “Bispecific Antibody by Protein Trans-splicing (BAPTS)”. Different from published methods, we assembled two parental antibody fragments in the hinge region by the protein trans-splicing reaction of a split intein to generate BsAbs without heavy/heavy and light/heavy chain mispairing. Utilizing this simple and efficient approach, there have been several BsAbs (CD3×HER2, CD3×EGFR, EGFR×HER2) synthesized to demonstrate its broad applicability. Correctly paired mAb arms were assembled to form BsAbs that were purified through protein A affinity chromatography to demonstrate industrial applicability at large scale. Further, the products were characterized through physical-biochemistry properties and biological activities to confirm expected quality of the products from “BAPTS”. More importantly, correct pairing was confirmed by mass spectrum. Proof-of-concept studies with CD3×HER2 BsAb (T-cell recruitment) demonstrated superior bioactivity compared with trastuzumab. The results of undetectable mispairing and high biological activity have indicated that this method has the potential to be utilized to manufacture BsAbs with high efficiency at industrial scale.

Background Prolactin receptor (PRLR) is highly expressed in a subset of human breast cancer and prostate cancer, which makes it a potential target for cancer treatment. In clinical trials, the blockade of PRLR was shown to be safe but with poor efficacy. It is therefore urgent to develop new therapies against PRLR target. Bispecific antibodies (BsAbs) could guide immune cells toward tumor cells, and produced remarkable effects in some cancers. Methods In this study, a bispecific antibody targeting both tumor antigen PRLR and T cell surface CD3 antigen (PRLR-DbsAb) was constructed by split intein mediated protein transsplicing (BAPTS) system for the first time. Its binding activity was determined by Biacore and Flow cytometry, and target-dependent T cell mediated cytotoxicity was detected using LDH release assay. ELISA was utilized to study the secretion of cytokines by immune cells. Subcutaneous tumor mouse models were used to analyze the in vivo anti-tumor effects of PRLR-DbsAb. Results PRLR-DbsAb in vitro could recruit and activate T cells to promote the release of Th1 cytokines IFN- γ and TNF- α , which could kill PRLR expressed breast cancer cells. In xenograft models with breast cancer cell line T47D, NOD/SCID mice intraperitoneally injected with PRLR-DbsAb exhibited significant inhibition of tumor growth and a longer survival compared to mice treated with PRLR monoclonal antibody (PRLR mAb). Conclusions Both in vitro and in vivo experiments showed PRLR-DbsAb had a potential therapy of cancer treatment potential therapy for cancer. Immunotherapy may be a promising treatment against the tumor target of PRLR.

Evapotranspiration (ET) is one of the important components of the global hydrologic cycle, energy exchange, and carbon cycle. However, basin scale actual ET (hereafter ETa) is difficult to estimate accurately. We present an evaluation of four actual ET products (hereafter ETp) in seven sub-basins in the Tibetan Plateau. The actual ET calculated by the water balance method (hereafter ETref) was used as the reference for correction of the different ETp. The ETref and ETp show obvious seasonal cycles, but the ETp overestimated or underestimated the ET of the sub-basins in the Tibetan Plateau. A simple and effective method was proposed to correct the basin-scale ETp. The method was referred to as ratio bias correction, and it can effectively remove nearly all biases of the ETp. The proposed method is simpler and more effective in correcting the four ETp compared with the gamma distribution bias correction method. The reliability of the ETp is significantly increased after the ratio bias correction. The ratio bias correction method was used to correct the ETp in the seven sub-basins in the Tibetan Plateau, and regional ET was significantly improved. The results may help improve estimation of the ET of the Tibetan Plateau and thereby contribute to a better understanding of the hydrologic cycle of the plateau.

Floodplain wetlands are of great importance in the entire river and floodplain ecosystems. Understanding the hydrological processes of floodplain wetlands is fundamental to study the changes in wetlands caused by climate change and human activities. In this study, floodplain wetlands along the middle reach of the Yellow River were selected as a study area. The hydrological processes and the interactions between the river and the underlying aquifer were investigated by combining remote sensing, hydraulic monitoring, and numerical modeling. Wetland areas from 2014 to 2019 were extracted from Landsat 8 remote sensing images, and their correlation with the river runoff was analyzed. The results indicate that the river flow had a limited impact on the wetland size and so did groundwater levels, due to the strong reliance of wetland vegetation on water levels. Based on hydrological and hydrogeological conditions, a surface water–groundwater coupled numerical model was established. The comparison and correlation analysis between the monitored groundwater head and the simulated river stage also show that river flow did not play a first-order role in controlling the groundwater levels of wetlands in the study area. The simulation results also suggest that it is the regional groundwater flow that mainly sustains shallow groundwater of floodplain wetlands in the study area. The floodplain wetland of the study area was dynamic zones between the regional groundwater and river, the contrasting pattern of hydrological regimes on both banks of the Yellow River was due to a combination of regional groundwater flow and topography.

Previous studies have examined free convection and the development of fingers in variable‐density groundwater environments, but the penetration rates of fingering processes (i.e., fingering speeds) have not been systematically investigated. Unlike common groundwater processes driven by advection and whose flow rates may be computed using Darcy's law, fingering speeds are far less intuitive. In this study, fingering speeds are analyzed in a natural convection system using two measurable diagnostics: deepest plume front (DPF, providing upper bounds on plume speeds) and vertical center of solute mass (COM, providing global speeds). The permeability, porosity, and dispersion (longitudinal and transverse dispersivities) were varied using a perturbation‐based stochastic approach to investigate their effects on fingering speeds. Modeling results show that the characteristic convective velocity, commonly used to represent theoretical fingering speeds, needs to incorporate effective porosity in a similar fashion to hydraulically driven average linear velocity and needs to be further adjusted by multiplying by a corrective factor f for predicting various fingering behaviors (approximately f = 0.115 for DPF and f = 0.034 for COM) in this study. A stochastic analysis demonstrates small variability in the time‐varying speed of both DPF and COM between model realizations. This indicates that reproducing fingering speeds is likely to be achieved and that one single realization can adequately produce f for the characteristic convective velocity. This study also identifies that f for speeds of DPF is most likely to be constrained by (0.115, 1.000), which is extremely useful in the design of laboratory and field experimentation. Key Points The effects of four hydrogeologic parameters on fingering speeds were examined A stochastic approach was used to study the variability of fingering speeds 3D effects on fingering speeds were briefly tested

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with angiotensin converting enzyme 2 (ACE2) interface, which enables 2G1 to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar half maximal inhibitory concentration in vitro. In SARS-CoV-2, Beta or Delta variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 is potentially capable of dealing with emerging SARS-CoV-2 variants in the future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.