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991.
Guillaume Terral Thierry Champion François Debaene Olivier Colas Maxime Bourguet Elsa Wagner-Rousset 《MABS-AUSTIN》2017,9(8):1317-1326
Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization. 相似文献
992.
Pharmacokinetic studies play an important role in all stages of drug discovery and development. Recent advancements in the tools for discovery and optimization of therapeutic proteins have created an abundance of candidates that may fulfill target product profile criteria. Implementing a set of in silico, small scale in vitro and in vivo tools can help to identify a clinical lead molecule with promising properties at the early stages of drug discovery, thus reducing the labor and cost in advancing multiple candidates toward clinical development. In this review, we describe tools that should be considered during drug discovery, and discuss approaches that could be included in the pharmacokinetic screening part of the lead candidate generation process to de-risk unexpected pharmacokinetic behaviors of Fc-based therapeutic proteins, with an emphasis on monoclonal antibodies. 相似文献
993.
Rajkumar Ganesan Ernest L. Raymond Detlev Mennerich Joseph R. Woska Jr. Gary Caviness Christine Grimaldi 《MABS-AUSTIN》2017,9(7):1143-1154
Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, β and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation. 相似文献
994.
Weichen Xu Rod Brian Jimenez Rachel Mowery Haibin Luo Mingyan Cao Nitin Agarwal 《MABS-AUSTIN》2017,9(7):1186-1196
During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective. 相似文献
995.
Nazzareno Dimasi Ryan Fleming Kris F. Sachsenmeier Binyam Bezabeh Carl Hay Jincheng Wu 《MABS-AUSTIN》2017,9(3):438-454
We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies. 相似文献
996.
Martin J. Scott Jennifer A. Lee Matthew S. Wake Kelly V. Batt Trevor A. Wattam Ian D. Hiles 《MABS-AUSTIN》2017,9(1):85-93
Bispecific antibodies (BsAbs) are emerging as an important class of biopharmaceutical. The majority of BsAbs are created from conventional antibodies or fragments engineered into more complex configurations. A recurring challenge in their development, however, is the identification of components that are optimised for inclusion in the final format in order to deliver both efficacy and robust biophysical properties. Using a modular BsAb format, the mAb-dAb, we assessed whether an ‘in-format’ screening approach, designed to select format-compatible domain antibodies, could expedite lead discovery. Human nerve growth factor (NGF) was selected as an antigen to validate the approach; domain antibody (dAb) libraries were screened, panels of binders identified, and binding affinities and potencies compared for selected dAbs and corresponding mAb-dAbs. A number of dAbs that exhibited high potency (IC50) when assessed in-format were identified. In contrast, the corresponding dAb monomers had ~1000-fold lower potency than the formatted dAbs; such dAb monomers would therefore have been omitted from further characterization. Subsequent stoichiometric analyses of mAb-dAbs bound to NGF, or an additional target antigen (vascular endothelial growth factor), suggested different target binding modes; this indicates that the observed potency improvements cannot be attributed simply to an avidity effect offered by the mAb-dAb format. We conclude that, for certain antigens, screening naïve selection outputs directly in-format enables the identification of a subset of format-compatible dAbs, and that this offers substantial benefits in terms of molecular properties and development time. 相似文献
997.
Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd’ and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation. 相似文献
998.
Human influence on the temporal dynamics and spatial distribution of forest biomass carbon in China 下载免费PDF全文
Weiwei Liu Fei Lu Yunjian Luo Wenjing Bo Lingqiao Kong Lu Zhang Bojie Liu Zhiyun Ouyang Xiaoke Wang 《Ecology and evolution》2017,7(16):6220-6230
Global carbon cycles are impacted by human activity primarily via fossil fuel combustion and forest carbon budget alterations. In this study, the temporal dynamics and spatial distribution of forest biomass carbon (FBC) stock and density in China were analyzed to assess the large‐scale effects of humans on FBC. The results indicated that from 1977 to 2013, the FBC stock increased by 62.9%, from 4,335 to 7,064 Tg C, owing to human‐driven forestation and ecological restoration programs. Because of intensive human impacts, 44.2%–54.6% of the FBC stock was concentrated in four provinces (Heilongjiang, Yunnan, Inner Mongolia, and Sichuan) and the FBC density increased from the densely populated southeastern provinces to the sparsely populated northeastern and western provinces. On a spatial scale, the FBC density was significantly negatively related to population density, and the degree of the dependence of the FBC density on population density has been declining since 1998. This improvement in human–forest relations is related to economic development and programs in China that have promoted forestation and reduced deforestation. These results suggest that human impacts, including forestation, deforestation, population density, and economic development, have played significant roles in determining the temporal and spatial variations of FBC in the anthropogenic era. Moreover, our findings have implications for forest management and improvement of the forest carbon sink in China. 相似文献
999.
Habitat selection by Eurasian lynx (Lynx lynx) is primarily driven by avoidance of human activity during day and prey availability during night 下载免费PDF全文
Marc Filla Joseph Premier Nora Magg Claudia Dupke Igor Khorozyan Matthias Waltert Luděk Bufka Marco Heurich 《Ecology and evolution》2017,7(16):6367-6381
The greatest threat to the protected Eurasian lynx (Lynx lynx) in Central Europe is human‐induced mortality. As the availability of lynx prey often peaks in human‐modified areas, lynx have to balance successful prey hunting with the risk of encounters with humans. We hypothesized that lynx minimize this risk by adjusting habitat choices to the phases of the day and over seasons. We predicted that (1) due to avoidance of human‐dominated areas during daytime, lynx range use is higher at nighttime, that (2) prey availability drives lynx habitat selection at night, whereas high cover, terrain inaccessibility, and distance to human infrastructure drive habitat selection during the day, and that (3) habitat selection also differs between seasons, with altitude being a dominant factor in winter. To test these hypotheses, we analyzed telemetry data (GPS, VHF) of 10 lynx in the Bohemian Forest Ecosystem (Germany, Czech Republic) between 2005 and 2013 using generalized additive mixed models and considering various predictor variables. Night ranges exceeded day ranges by more than 10%. At night, lynx selected open habitats, such as meadows, which are associated with high ungulate abundance. By contrast, during the day, lynx selected habitats offering dense understorey cover and rugged terrain away from human infrastructure. In summer, land‐cover type greatly shaped lynx habitats, whereas in winter, lynx selected lower altitudes. We concluded that open habitats need to be considered for more realistic habitat models and contribute to future management and conservation (habitat suitability, carrying capacity) of Eurasian lynx in Central Europe. 相似文献
1000.