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Jianlin Xu, Peifeng Tang, Andrew Yongky, Barry Drew, Michael C Borys, Shijie Liu, Zheng Jian Li
Temperature shift (TS) to a hypothermic condition has been widely used during protein production processes that use Chinese hamster ovary (CHO) cells. The effect of temperature on cell growth, metabolites, protein titer and quality depends on cell line, product, and other bioreactor conditions. Due to the large numbers of experiments, which typically last 2-3 weeks each, limited systematic TS studies have been reported with multiple shift temperatures and steps at different times. Here, we systematically studied the effect of temperature on cell culture performance for the production of two monoclonal antibodies by industrial GS and DG44 CHO cell lines...
September 19, 2018: MAbs
Yutong Jin, Ziqing Lin, Qingge Xu, Cexiong Fu, Zhaorui Zhang, Qunying Zhang, Wayne A Pritts, Ying Ge
The pharmaceutical industry's interest in monoclonal antibodies (mAbs) and their derivatives has spurred rapid growth in the commercial clinical pipeline of these effective therapeutics. The complex micro-heterogeneity of mAbs requires in-depth structural characterization for critical quality attribute assessment and quality assurance. Currently, mass spectrometry (MS)-based methods are the gold standard in mAb analysis, primarily with a bottom-up approach in which immunoglobulins G (IgGs) and their variants are digested into peptides to facilitate the analysis...
September 19, 2018: MAbs
Heather Cooke, Joe Arndt, Chao Quan, Renée I Shapiro, Dingyi Wen, Susan Foley, Malgorzata M Vecchi, Martin Preyer
Bispecific antibody therapeutics can expand the functionality of a conventional monoclonal antibody drug because they can bind multiple antigens. However, their great potential is counterbalanced by the challenges faced in their production. The classic asymmetric bispecific containing an Fc requires the expression of four unique chains - two light chains and two heavy chains; each light chain must pair with its correct heavy chain, which then must heterodimerize to form the full bispecific. The light-chain pairing problem has several solutions, some of which require engineering and optimization for each bispecific pair...
September 14, 2018: MAbs
Jun Zhao, Ruth Nussinov, Buyong Ma
A key question in immunology is whether antigen recognition and Fc receptor (FcR) binding are allosterically linked. This question is also relevant for therapeutic antibody design. Antibody Fab and Fc domains are connected by flexible unstructured hinge region. Fc chains have conserved glycosylation sites at Asn297, with each conjugated to a core heptasaccharide and forming biantennary Fc glycan. The glycans modulate the Fc conformations and functions. It is well known that the antibody Fab and Fc domains and glycan affect antibody activity, but whether these elements act independently or synergistically is still uncertain...
September 13, 2018: MAbs
Shouheng Lin, Guohua Huang, Lin Cheng, Zhen Li, Yiren Xiao, Qiuhua Deng, Yuchuan Jiang, Baiheng Li, Simiao Lin, Suna Wang, Qiting Wu, Huihui Yao, Su Cao, Yang Li, Pentao Liu, Wei Wei, Duanqing Pei, Yao Yao, Zhesheng Wen, Xuchao Zhang, Yilong Wu, Zhenfeng Zhang, Shuzhong Cui, Xiaofang Sun, Xueming Qian, Peng Li
Animal models used to evaluate efficacies of immune checkpoint inhibitors are insufficient or inaccurate. We thus examined two xenograft models used for this purpose, with the aim of optimizing them. One method involves the use of peripheral blood mononuclear cells and cell line-derived xenografts (PBMCs-CDX model). For this model, we implanted human lung cancer cells into NOD-scid-IL2Rg-/- (NSI) mice, followed by injection of human PBMCs. The second method involves the use of hematopoietic stem and progenitor cells and CDX (HSPCs-CDX model)...
September 11, 2018: MAbs
Nobuo Sekiguchi, Chiyomi Kubo, Ayako Takahashi, Kumiko Muraoka, Akira Takeiri, Shunsuke Ito, Mariko Yano, Futa Mimoto, Atsuhiko Maeda, Yuki Iwayanagi, Tetsuya Wakabayashi, Shotaro Takata, Naoaki Murao, Shuichi Chiba, Masaki Ishigai
Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process...
September 10, 2018: MAbs
Danielle Mandikian, Hanine Rafidi, Pragya Adhikari, Priya Venkatraman, Lidia Nazarova, Gabriel Fung, Isabel Figueroa, Gregory Z Ferl, Sheila Ulufatu, Jason Ho, Cynthia McCaughey, Jeffrey Lau, Shang-Fan Yu, Saileta Prabhu, Jack Sadowsky, C Andrew Boswell
Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder 'click' reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components...
September 10, 2018: MAbs
Chao Zheng, Joshuaine Toth, Tammy Bigwarfe, Margit MacDougall, Kavita Jerath, Kristin Bovat, James Smith, Peng Sun, David Hayes, Ryan Fryer, Sanjaya Singh, Rachel Kroe-Barrett
Ang1 is a soluble ligand to receptor Tie2, and increasing the circulating Ang1 level may improve vascular stabilization under certain disease conditions. Here, we found that the circulating Ang1 level was significantly increased in cynomolgus monkeys treated with non-neutralizing anti-Ang1 antibodies. Improving the antibodies' pharmacokinetic properties by IgG Fc mutations further increased the circulating Ang1 level. However, the mutations decreased the thermal stability of the molecules, which may limit their use as therapeutic antibodies...
September 10, 2018: MAbs
Zvezdan Pavlovic, Jarrett J Adams, Levi L Blazer, Amandeep K Gakhal, Nick Jarvik, Zachary Steinhart, Mélanie Robitaille, Keith Mascall, James Pan, Stephane Angers, Jason Moffat, Sachdev S Sidhu
Secreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8...
September 5, 2018: MAbs
Douglas D Leipold, Isabel Figueroa, Shabkhaiz Masih, Brandon Latifi, Victor Yip, Ben-Quan Shen, Randall C Dere, Montserrat Carrasco-Triguero, M Violet Lee, Ola M Saad, Luna Liu, Jintang He, Dian Su, Keyang Xu, Brian R Vuillemenot, Steven T Laing, Melissa Schutten, Katherine R Kozak, Bing Zheng, Andrew G Polson, Amrita V Kamath
Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker...
September 5, 2018: MAbs
Chunlei Wang, Bhargavi Vemulapalli, Mingyan Cao, Dhanesh Gadre, Jihong Wang, Alan Hunter, Xiangyang Wang, Dengfeng Liu
Immunoglobulin G-like bispecific antibodies with asymmetric architecture are among the most widely used bispecific antibody formats for diagnostic and therapeutic applications. The primary technical challenge for this format is how to achieve correctly paired assembly of four unique polypeptide chains. Advances in protein engineering and process development are being used to overcome these challenges and are driving a corresponding demand for sensitive analytical tools to monitor and control mispaired species...
August 28, 2018: MAbs
Christopher S Spahr, Mark E Daris, Kevin C Graham, Brian D Soriano, Jennitte L Stevens, Stone D-H Shi
Protein-based biotherapeutics are produced in engineered cells through complex processes and may contain a wide variety of variants and post-translational modifications that must be monitored or controlled to ensure product quality. Recently, a low level (~1-5%) impurity was observed in a number of proteins derived from stably transfected Chinese hamster ovary (CHO) cells using mass spectrometry. These molecules include antibodies and Fc fusion proteins where Fc is on the C-terminus of the construct. By liquid chromatography-mass spectrometry (LC-MS), the impurity was found to be ~1177 Da larger than the expected mass...
August 27, 2018: MAbs
Brian M Maas, Yanguang Cao
Manipulation of binding affinity between monoclonal antibodies (mAbs) and the neonatal Fc receptor (FcRn) has been leveraged to extend mAb half-life; however, the steps required for success remain ambiguous and experimental observations are inconsistent. Recent models have considered the time course of endosomal transit a major contributor to the relationship between FcRn affinity and antibody half-life. Our objective was to develop a minimal physiologically based pharmacokinetic model to explain how changes in IgG-FcRn association rate constant (Kon ), dissociation rate constant (Koff ), and endosomal transit time [T(w) ] translate to improved IgG clearance across mice, monkeys and humans...
August 21, 2018: MAbs
Mingyan Cao, Chunlei Wang, Wai Keen Chung, Dana Motabar, Jihong Wang, Elizabeth Christian, Shihua Lin, Alan Hunter, Xiangyang Wang, Dengfeng Liu
Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development...
August 21, 2018: MAbs
Yukinori Endo, Yi Shen, Lamis Abou Youssef, Nishant Mohan, Wen Jin Wu
Ado-trastuzumab emtansine (Kadcyla®; T-DM1) is an antibody-drug conjugate developed to treat trastuzumab-resistant disease. Despite initial favorable outcomes, most patients eventually cease to respond due to developing acquired resistance to T-DM1. Currently, there is no targeted therapy to treat T-DM1-resistant disease. To explore novel therapeutic targets to improve therapeutic efficacy of T-DM1, we generated T-DM1-resistant cells using trastuzumab-resistant JIMT1 cells. We found that the loss of human epidermal growth factor receptor 2 confers T-DM1 resistance, which in turn activates a compensatory mechanism that increases epidermal growth factor receptor (EGFR) expression...
August 21, 2018: MAbs
Danielle M DiCara, Nisana Andersen, Ruby Chan, James A Ernst, Gai Ayalon, Greg A Lazar, Nicholas J Agard, Amy Hilderbrand, Isidro Hötzel
Developability assessment of therapeutic antibody candidates assists drug discovery by enabling early identification of undesirable instabilities. Rapid chemical stability screening of antibody variants can accelerate the identification of potential solutions. We describe here the development of a high-throughput assay to characterize asparagine deamidation. We applied the assay to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity...
August 21, 2018: MAbs
Jing Zhen, John Kim, Ying Zhou, Ervinas Gaidamauskas, Shyamsundar Subramanian, Ping Feng
Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) is a technique that is increasingly being used as a trapping/enrichment tool for glycopeptides/phosphorylated peptides or sample fractionation in proteomics research. Here, we describe a novel ERLIC-MS/MS-based peptide mapping method that was successfully used for the characterization of denosumab, in particular the analysis of sequence coverage, terminal peptides, methionine oxidation, asparagine deamidation and glycopeptides...
August 21, 2018: MAbs
Bhumit A Patel, Adrian Gospodarek, Michael Larkin, Sophia A Kenrick, Mark A Haverick, Nihal Tugcu, Mark A Brower, Douglas D Richardson
For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, Mw and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset Mw criteria are met or unmet...
August 21, 2018: MAbs
Bryan Smith, Andrea Kiessling, Rocio Lledo-Garcia, Kate L Dixon, Louis Christodoulou, Matthew C Catley, Paul Atherfold, Lena E D'Hooghe, Helene Finney, Kevin Greenslade, Hanna Hailu, Lara Kevorkian, Daniel Lightwood, Christoph Meier, Rebecca Munro, Omar Qureshi, Kaushik Sarkar, Sophie P Shaw, Roohi Tewari, Alison Turner, Kerry Tyson, Shauna West, Stevan Shaw, Frank R Brennan
Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin)...
August 21, 2018: MAbs
Anneleen Steels, Adriaan Verhelle, Olivier Zwaenepoel, Jan Gettemans
The tumor suppressor p53 is of crucial importance in the prevention of cellular transformation. In the presence of cellular stress signals, the negative feedback loop between p53 and Mdm2, its main negative regulator, is disrupted, which results in the activation and stabilization of p53. Via a complex interplay between both transcription-dependent and - independent functions of p53, the cell will go through transient cell cycle arrest, cellular senescence or apoptosis. However, it remains difficult to completely fathom the mechanisms behind p53 regulation and its responses, considering the presence of multiple layers involved in fine-tuning them...
August 15, 2018: MAbs
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