Glycoengineering of CHO cells for production of recombinant therapeutics with enhanced efficacy

Glycosylation can significantly affect the efficacy of recombinant therapeutics. Glycoprotein drugs, such as EPO, require a high degree of sialylation on their N-glycans in order to have a longer circulatory half-life. Mannose-terminated N-glycans can target the protein to dendritic cells and macrophages cells via cell surface mannose-binding receptors. Removal of core fucose from human IgG1 antibodies has been shown to significantly enhance its affinity to FcRIIIa and thereby dramatically improves its antibody-dependent cellular cytotoxicity (ADCC). Cancer cells generally express glycoproteins with shortened O-glycans. Therefore, recombinant anti-cancer vaccines carrying these short tumor-associated O-glycans are more ideal for triggering specific anti-tumor immune responses. With cytotoxic lectins and genome editing tools, namely ZFNs, TALENs and CRISPR-Cas9, we have so far created 29 CHO glycosylation mutant cell lines (CHO-gmt1 to CHO-gmt29; gmt = glycosylation mutant). In some of these mutant lines, only one gene has been inactivated (CHO-gmt1, 2, 3 4 cells), whereas in others more than 10 glycosylation genes have been knocked out in order to obtain a particular glycan structure. With these mutants, we have been able to produce EPO with highly sialylated N-glycans for extending in vivo half-life. We have developed stable cell lines to produce recombinant human β-glucocerebrosidase with mannose-terminated N-glycans (mainly Man5). This product is similar to Cerezyme, but the in vitro glycan modification is not needed. We have also developed stable cell lines to produce fucose-free rituximab and GA101 with high titers. In a cell-based ADCC assay, these fucose-free rituximab and GA101 outperformed their commercial counterparts, Rituxan and Gazyva, respectively. Furthermore, rituximab produced by more than a dozen of these CHO-gmts were purified and the glycans attached to the antibody were analyzed by MALDI-TOF MS and LC-MS. The results showed that many of these mutants were able to produce the antibody with one major N-glycan which represents 90-97% of the total N-glycans attached to the antibody. These antibodies are invaluable tools for studying the impact of glycans on antibody functions including PK/PD characteristics. Rituximab produced by these CHO-gmt cells have been compared in a cell-based ADCC assay. Their binding affinities for FcRII, FcRIIIa and the neonatal Fc receptor (FcRn) were also determined. As expected, fucose-free glycans bind FcRIIIa much tighter than others, and are most effective in activating ADCC. While most glycans did not affect the binding to FcRII and FcRn, two glycans significantly enhanced the binding for FcRII and FcRn. The in vivo cell killing ability and circulating half-life for the rituximab antibodies that carry different N-glycans are being investigated in mouse models. Taken together, we have generated a large panel of CHO glycosylation mutants. Each of these mutants produces a unique set of N- and O-glycans. They can be used to study the impacts of glycans on therapeutic efficacies of biologics. Our study on the rituximab carrying different homogeneous N-glycans represents a first systematic investigation on the impact of glycans on antibody functions

Segmentation method of U-net sheet metal engineering drawing based on CBAM attention mechanism

In the manufacturing process of heavy industrial equipment, the specific unit in the welding diagram is first manually redrawn and then the corresponding sheet metal parts are cut, which is inefficient. To this end, this paper proposes a U-net-based method for the segmentation and extraction of specific units in welding engineering drawings. This method enables the cutting device to automatically segment specific graphic units according to visual information and automatically cut out sheet metal parts of corresponding shapes according to the segmentation results. This process is more efficient than traditional human-assisted cutting. Two weaknesses in the U-net network will lead to a decrease in segmentation performance: first, the focus on global semantic feature information is weak, and second, there is a large dimensional difference between shallow encoder features and deep decoder features. Based on the CBAM (Convolutional Block Attention Module) attention mechanism, this paper proposes a U-net jump structure model with an attention mechanism to improve the network's global semantic feature extraction ability. In addition, a U-net attention mechanism model with dual pooling convolution fusion is designed, the deep encoder's maximum pooling + convolution features and the shallow encoder's average pooling + convolution features are fused vertically to reduce the dimension difference between the shallow encoder and deep decoder. The dual-pool convolutional attention jump structure replaces the traditional U-net jump structure, which can effectively improve the specific unit segmentation performance of the welding engineering drawing. Using vgg16 as the backbone network, experiments have verified that the IoU, mAP, and Accu of our model in the welding engineering drawing dataset segmentation task are 84.72%, 86.84%, and 99.42%, respectively

Research on Online Discourse of Cross-Border E-commerce Platform on the Basis of Co-operative Principle

China’s cross-border e-commerce industry has shown a blow-out trend fueled by the rapidly-improved informatization of the society and powerful support of government. However, the increasing platforms in the market, the homogenizing merchandise, and the decreasing of conversion cost bring enormous challenges to the platforms. In order to survive in the fierce competition, they must improve the quality of services in the communication with consumers so as to enhance the core competitiveness centered on consumers. In this research, we compiled 100 real cases of cross-border e-commerce platform online discourse by collecting chat records from the online interaction between 50 college students and the platform merchants on the Tmall international platform. This study analyzes these online discourse cases from the perspective of Grice’s Co-operative principle. The study demonstrates that, the application of Grice’s Co-operative principle can enable cross-border e-commerce platforms to effectively adopt and employ the most appropriate interactive discourse, which exerts an tremendous influence on improving the quality of communication services. And there is a positive correlation between the usage of the Co-operative principle and the monthly sales volume of the platform

Mutually-orthogonal unitary and orthogonal matrices

We introduce the concept of n-OU and n-OO matrix sets, a collection of n mutually-orthogonal unitary and real orthogonal matrices under Hilbert-Schmidt inner product. We give a detailed characterization of order-three n-OO matrix sets under orthogonal equivalence. As an application in quantum information theory, we show that the minimum and maximum numbers of an unextendible maximally entangled bases within a real two-qutrit system are three and four, respectively. Further, we propose a new matrix decomposition approach, defining an n-OU (resp. n-OO) decomposition for a matrix as a linear combination of n matrices from an n-OU (resp. n-OO) matrix set. We show that any order-d matrix has a d-OU decomposition. As a contrast, we provide criteria for an order-three real matrix to possess an n-OO decomposition.Comment: 16 pages, no figur