Search

Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling. [Display omitted] • IgGs against the snake venom toxins α-cobratoxin and myotoxin II were discovered • Light-chain shuffling was used to generate IgGs with pH-dependent binding properties • Binding between IgG and snake venom toxins affects FcRn-mediated cellular handling • Cellular handing of IgG-bound toxins is complex and affected by multiple factors Tulika et al. utilized light-chain shuffling and phage display technology to generate IgG1 antibodies with pH-dependent antigen binding properties against snake venom toxins. Combined with Fc-engineering, this strategy may be useful for the development of acid-switched antibodies. [ABSTRACT FROM AUTHOR]

Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N‐terminal Gly‐His6 segment. This tag promoted acylation of the N‐terminal Nα‐amine resulting in stable conjugates. Herein, we report the peptide sequences Hisn‐Lys‐Hism, which we term Lys‐His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys Nϵ‐amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys‐His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C‐terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys‐His tag acylation method. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cell lines are widely used in industry for biological drug production. During cell culture development, considerable effort is invested to understand the factors that greatly impact cell growth, specific productivity and product qualities of the biotherapeutics. While high-throughput omics approaches have been increasingly utilized to reveal cellular mechanisms associated with cell line phenotypes and guide process optimization, comprehensive omics data analysis and management have been a challenge. Here we developed CHOmics, a web-based tool for integrative analysis of CHO cell line omics data that provides an interactive visualization of omics analysis outputs and efficient data management. CHOmics has a built-in comprehensive pipeline for RNA sequencing data processing and multi-layer statistical modules to explore relevant genes or pathways. Moreover, advanced functionalities were provided to enable users to customize their analysis and visualize the output systematically and interactively. The tool was also designed with the flexibility to accommodate other types of omics data and thereby enabling multi-omics comparison and visualization at both gene and pathway levels. Collectively, CHOmics is an integrative platform for data analysis, visualization and management with expectations to promote the broader use of omics in CHO cell research. Author summary: Recombinant proteins have dominated recent blockbuster therapeutic drugs, accounting for 11 of the top 15 drugs by sales. Chinese hamster ovary (CHO) cells are the most widely used expression system for biomanufacturing of many of these biotherapies. Thus, there is increasing interest in leveraging omics technologies for CHO cell line development, bioprocess optimization, and biotherapeutic product quality assessment. However, CHO cells have been largely ignored in the development of publicly available tools to facilitate comprehensive omics data analysis and management, despite being a ubiquitous research tool and biotherapeutic production host. To address the gap, we have recently developed a web-based tool, named "CHOmics", for the integrative and interactive data analysis and visualization specifically designed for CHO. This novel tool provides all-in-one solutions from raw data processing to pathway and gene analysis and offers considerable flexibility to customize analysis and visualization. It further allows for other omics data inputs and thereby enables multi-omics comparison. The open-source tool is freely available at http://www.chomics.org. [ABSTRACT FROM AUTHOR]

Achieving the predictable expression of heterologous genes in a production host has proven difficult. Each heterologous gene expressed in the same host seems to elicit a different host response governed by unknown mechanisms. Historically, most studies have approached this challenge by manipulating the properties of the heterologous gene through methods like codon optimization. Here we approach this challenge from the host side. We express a set of 45 heterologous genes in the same Escherichia coli strain, using the same expression system and culture conditions. We collect a comprehensive RNAseq set to characterize the host's transcriptional response. Independent Component Analysis of the RNAseq data set reveals independently modulated gene sets (iModulons) that characterize the host response to heterologous gene expression. We relate 55% of variation of the host response to: Fear vs Greed (16.5%), Metal Homeostasis (19.0%), Respiration (6.0%), Protein folding (4.5%), and Amino acid and nucleotide biosynthesis (9.0%). If these responses can be controlled, then the success rate with predicting heterologous gene expression should increase. • Transcriptomics during expression of 40 heterologous proteins. • 4 major host cell responses to heterologous protein expression. • Expression levels are negatively correlated with stress response. • Metal homeostasis and respiration affected during heterologous protein expression. • Amino acids show trade-offs between their synthesis cost, usage and adaptivity. [ABSTRACT FROM AUTHOR]

Despite the considerable global impact of snakebite envenoming, available treatments remain suboptimal. Here, we report the discovery of a broadly-neutralizing human monoclonal antibody, using a phage display-based cross-panning strategy, capable of reducing the cytotoxic effects of venom phospholipase A 2 s from three different snake genera from different continents. This highlights the potential of utilizing monoclonal antibodies to develop more effective, safer, and globally accessible polyvalent antivenoms that can be widely used to treat snakebite envenoming. [Display omitted] • Improved and more accessible snakebite therapies are needed to reduce the negative effects caused by snakebite envenoming. • Recombinant antivenoms, composed of monoclonal antibodies have been hypothesized as a future snakebite envenoming therapy. • A novel human monoclonal IgG antibody shows broadly-neutralizing capabilities against myotoxicity from viperid snake venoms. • This broad neutralization affects venom from snakes belonging to three genera and originating from three continents. • While cross-reactive, the antibody retains selectivity to bind only a subset of the tested viperid venoms. [ABSTRACT FROM AUTHOR]