Structural Model for the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2

Epa, V. Chandana; Dolezal, Olan; Doughty, Larissa; Xiao, Xiaowen; Jöst, Christian; Plückthun, Andreas; Adams, Timothy E.

doi:10.1371/journal.pone.0059163

Cited by 19 publications

(17 citation statements)

References 35 publications

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“…Importantly, we observed that 111 In-(HE) 3 -G3 was able to bind to HER2 in the presence of trastuzumab. This confirms the results of structural modelling which demonstrate that DARPin G3 and trastuzumab bind to non-overlapping epitopes of HER2 domain IV [ 35 ]. Thus, 111 In-(HE) 3 -G3 DARPin has the potential to image both treatment-naive patients and patients receiving concomitant trastuzumab without requiring a delay to treatment.…”

Section: Discussionsupporting

confidence: 88%

Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging

Goldstein

Sosabowski

Livanos

et al. 2014

Eur J Nucl Med Mol Imaging

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PurposeHuman epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.MethodsG3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with 125I, or with 111In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts.ResultsFor both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from 111In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from 125I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, 111In-labelled and 125I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with 111In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours.ConclusionRadiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-014-2940-2) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 88%

Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging

Goldstein

Sosabowski

Livanos

et al. 2014

Eur J Nucl Med Mol Imaging

Self Cite

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“…[68]) and binding hotspots for drug design [25], altering binding kinetics [13], [64], protein-protein docking (e.g. [19]), and characterising transition states (e.g. [75]), binding pathways [61], and sequence-affinity landscapes [2].…”

Section: Introductionmentioning

confidence: 99%

SKEMPI 2.0: An updated benchmark of changes in protein-protein binding energy, kinetics and thermodynamics upon mutation

Jankauskaite

Jiménez‐García

Dapkūnas

et al. 2018

Preprint

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Motivation: Understanding the relationship between the sequence, structure, binding energy, binding kinetics and binding thermodynamics of protein-protein interactions is crucial to understanding cellular signaling, the assembly and regulation of molecular complexes, the mechanisms through which mutations lead to disease, and protein engineering. Results: We present SKEMPI 2.0, a major update to our database of binding free energy changes upon mutation for structurally resolved protein-protein interactions. This version now contains manually curated binding data for 7085 mutations, an increase of 133%, including changes in kinetics for 1844 mutations, enthalpy and entropy changes for 443 mutations, and 440 mutations which abolish detectable binding. Availability:The database is available at

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“…2 to create the phagemid pGBid Trast Fab. M13 phage particles produced with SS320 E. coli cells carrying pGBid Trast Fab or the insert-free parental phagemid pGBid were evaluated for binding to immobilized HER2 extracellular domain 21 . As shown in Fig.…”

Section: Phage Display Of a Her2-binding Fab Using Gbidmentioning

confidence: 99%

“…(2020) 10:2888 | https://doi.org/10.1038/s41598-020-59745-2 www.nature.com/scientificreports www.nature.com/scientificreports/ Phage ELISA to evaluate HER2 binding. The soluble extracellular domain of HER2 (ErbB2) was a gift from Prof. Timothy Adams of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) 21 . Nunc MaxiSorp uncoated ELISA plates (BioLegend) were coated overnight at 4 °C with 100 μL of either ErbB2 (2 μg/mL in DPBS) or DPBS.…”

Section: Scientific Reports |mentioning

confidence: 99%

Golden Gate assembly with a bi-directional promoter (GBid): A simple, scalable method for phage display Fab library creation

Chockalingam

Peng

Vuong

et al. 2020

Sci Rep

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Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method -Golden Gate assembly with a bidirectional promoter (GBid) -for constructing phage display fab libraries. in GBid, the constant domains of the Fabs are located in the backbone of the phagemid vector and the library insert comprises only the variable regions of the antibodies and a central bi-directional promoter. This vector design reduces the process of Fab library construction to "scFv-like" simplicity and the double promoter ensures robust expression of both constituent chains. To maximize the library size, the 3 fragments comprising the insert -two variable chains and one bi-directional promoter -are assembled via a 3-fragment overlap extension PCR and the insert is incorporated into the vector via a high-efficiency one-fragment, one-pot Golden Gate assembly. the reaction setup requires minimal preparatory work and enzyme quantities, making GBid highly scalable. Using GBid, we constructed a chimeric chicken-human fab phage display library comprising 10 10 variants targeting the multi-transmembrane protein human CD20 (hCD20). Selection/counter-selection on transfected whole cells yielded hCD20-specific antibodies in four rounds of panning. The simplicity and scalability of GBid makes it a powerful tool for the discovery/engineering of fabs and igGs.The discovery and engineering of monoclonal antibodies (mAbs) using phage display usually utilizes one of two simplified display formats. Single-chain variable fragments (scFvs) represent the simplest format for antibody phage display in which only the domains directly involved in interaction with the antigen -the variable heavy and variable light chains -are represented. Antibody-binding fragments (Fabs) more closely resemble the antigen-binding "business end" of IgG molecules and incorporate both the variable regions and their immediately neighboring constant subdomain -typically CH1 for the heavy chain and CLκ or CLλ for the light chain. Under oxidizing conditions, the constant domains in Fabs are covalently linked via a disulfide bond.The attractiveness of the scFv as a medium for IgG discovery lies in its overall simplicity. The presence of only a single chain generally gives rise to higher expression, and cloning of scFvs into phagemid vectors is relatively straightforward, typically requiring only a single overlap extension PCR reaction that links the two variable domains via a short flexible linker 1-3 . Unfortunately, the binding affinity of scFvs can change significantly upon reformatting to Fabs or IgGs, creating a need to verify the binding/activity of the molecules in the more complex formats in applications where Fabs or IgGs are the desired final product 4-8 .Fabs more closely resemble full-length IgGs and are generally more stable than scFvs 6,9 , making these molecules a more reliable indicator of full-length IgG behavior. Unfortunately, the construction of phage display Fab libraries is less straight...

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Structural Model for the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2

Cited by 19 publications

References 35 publications

Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging

Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging

SKEMPI 2.0: An updated benchmark of changes in protein-protein binding energy, kinetics and thermodynamics upon mutation

Golden Gate assembly with a bi-directional promoter (GBid): A simple, scalable method for phage display Fab library creation

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