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Find video protocols related to scientific articles indexed in Pubmed.
High-throughput epitope binning assays on label-free array-based biosensors can yield exquisite epitope discrimination that facilitates the selection of monoclonal antibodies with functional activity.
PLoS ONE
PUBLISHED: 01-01-2014
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Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending upon the sample availability, and the number and diversity of the interactions being studied.
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A therapeutic anti-VEGF antibody with increased potency independent of pharmacokinetic half-life.
Cancer Res.
PUBLISHED: 03-30-2010
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Bevacizumab [Avastin; anti-vascular endothelial growth factor (VEGF) antibody] is an antiangiogenic IgG approved for treating patients with certain types of colon, breast, and lung cancer. In these indications, bevacizumab is administered every 2 to 3 weeks, prompting us to study ways to reduce the frequency of administration. Increasing affinity to neonatal Fc receptor (FcRn) may extend the pharmacokinetic half-life of an antibody, but the quantitative effect of FcRn affinity on clearance has not been clearly elucidated. To gain further insight into this relationship, we engineered a series of anti-VEGF antibody variants with minimal amino acid substitutions and showed a range of half-life improvements in primates. These results suggest that, if proven clinically safe and effective, a modified version of bevacizumab could potentially provide clinical benefit to patients on long-term anti-VEGF therapy through less-frequent dosing and improved compliance with drug therapy. Moreover, despite having half-life similar to that of wild-type in mice due to the species-specific FcRn binding effects, the variant T307Q/N434A exhibited superior in vivo potency in slowing the growth of certain human tumor lines in mouse xenograft models. These results further suggest that FcRn variants may achieve increased potency through unidentified mechanisms in addition to increased systemic exposure.
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Engineering human IgG1 affinity to human neonatal Fc receptor: impact of affinity improvement on pharmacokinetics in primates.
J. Immunol.
PUBLISHED: 06-05-2009
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The pH-dependent binding of Igs to the neonatal FcR (FcRn) plays a critical role in the in vivo homeostasis of IgGs. Modulating the interaction between Fc and FcRn through protein engineering is one method for improving the pharmacokinetics of therapeutic Abs. Recent studies disputed the direct relationship between increasing FcRn affinity and improved pharmacokinetic properties. In this work, we studied the pharmacokinetics of two human IgG1 Fc variants in cynomolgus monkey to further clarify the affinity-pharmacokinetic relationship. First, we report a number of novel Fc point mutations and combination variants, including some with primate-specific FcRn-binding improvements. By studying these variants along with some previously described variants across a wide range of affinities, we discovered a direct correlation of pH 6 affinity improvements with neutral pH improvements, suggesting that all of the tested variants exhibit similar pH dependency in FcRn binding. We then evaluated the pharmacokinetics of variants N434A and N434W, which, respectively, gave approximately 4- and 80-fold improvements in pH 6-binding affinity to both human and nonhuman primate FcRn. Surprisingly, clearance of N434W was similar to that of wild type. N434W is the first variant studied in primates that exhibits significant binding to FcRn at pH 7.4, and its clearance substantiates the principle that too much affinity improvement, i.e., beyond that of N434W, does not yield improved pharmacokinetics. In contrast, N434A exhibited a approximately 2-fold decrease in clearance in cynomolgus monkey, supporting the notion that modest increases in pH 6 FcRn affinity can result in improved pharmacokinetics in primates.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.