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High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement.

ABSTRACT: Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudomonas aeruginosa PcrV. PcrV is an essential part of the P. aeruginosa type III secretion system (T3SS), and its oligomeric nature allows for multiple complex binding and blocking options. The library screening yielded a large number of promising biparatopic lead candidates, revealing significant (and non-trivial) preferences in terms of Nanobody building block and epitope bin combinations and orientations. Excellent potencies were confirmed upon further characterization in two different P. aeruginosa T3SS-mediated cytotoxicity assays. Three biparatopic Nanobodies were evaluated in a lethal mouse P. aeruginosa challenge pneumonia model, conferring 100% survival upon prophylactic administration and reducing lung P. aeruginosa burden by up to 2 logs. At very low doses, they protected the mice from P. aeruginosa infection-related changes in lung histology, myeloperoxidase production, and lung weight. Importantly, the most potent Nanobody still conferred protection after therapeutic administration up to 24 h post-infection. The concept of screening such formatting libraries for potency improvement is applicable to other targets and biological therapeutic platforms.

SUBMITTER: De Tavernier E

PROVIDER: S-EPMC4946937 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement.

De Tavernier Evelyn E Detalle Laurent L Morizzo Erika E Roobrouck Annelies A De Taeye Severine S Rieger Melanie M Verhaeghe Tom T Correia Andreia A Van Hegelsom Rob R Figueirido Rita R Noens Jeroen J Steffensen Søren S Stöhr Thomas T Van de Velde Willem W Depla Erik E Dombrecht Bruno B

The Journal of biological chemistry 20160520 29

Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudomonas aeruginosa PcrV. PcrV is an essential part o ...[more]

PMID: 27226529

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Project description:We present a novel method utilizing "saltless" pH gradient weak cation exchange-hydrophilic interaction liquid chromatography directly coupled to electron transfer dissociation (ETD) mass spectrometry for the automated on-line high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resolution mass spectrometer, displays an improvement over existing methods with an approximately 100-fold reduction in sample requirements and analysis time. The scheme presented is capable of identifying all of the major combinatorial histone codes present in a sample in a 2-h analysis. The large N-terminal histone peptides are eluted by the pH and organic solvent weak cation exchange-hydrophilic interaction liquid chromatography gradient and directly introduced via nanoelectrospray ionization into a benchtop linear quadrupole ion trap mass spectrometer equipped with ETD. Each polypeptide is sequenced, and the modification sites are identified by ETD fragmentation. The isobaric trimethyl and acetyl modifications are resolved chromatographically and confidently distinguished by the synthesis of mass spectrometric and chromatographic information. We demonstrate the utility of the method by complete characterization of human histone H3.2 and histone H4 from butyrate-treated cells, but it is generally applicable to the analysis of highly modified peptides. We find this methodology very useful for chromatographic separation of isomeric species that cannot be separated well by any other chromatographic means, leading to less complicated tandem mass spectra. The improved separation and increased sensitivity generated novel information about much less abundant forms. In this method demonstration we report over 200 H3.2 forms and 70 H4 forms, including forms not yet detected in human cells, such as the remarkably highly modified histone H3.2 K4me3K9acK14acK18acK23acK27acK36me3. Such detail provided by our proteomics platform will be essential for determining how histone modifications occur and act in combination to propagate the histone code during transcriptional events and could greatly enable sequencing of the histone component of human epigenomes.

Dataset Information

High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement.

Publications

High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement.

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