Directed Evolution to Low Nanomolar Affinity of a Tumor-Targeting Epidermal Growth Factor Receptor-Binding Affibody Molecule

Friedman, Mikaela; Orlova, Anna; Johansson, E.; Eriksson, Tove; Höidén-Guthenberg, Ingmarie; Tolmachev, Vladimir; Nilsson, Fred; Ståhl, Stefan

doi:10.1016/j.jmb.2007.12.060

Cited by 139 publications

(169 citation statements)

References 51 publications

(59 reference statements)

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“…The targeted tracer used in this experiment was Affibody anti-EGFR imaging agent (Affibody) conjugated to Licor IRDye 800CW fluorescent dye. This anti-EGFR imaging agent is a three-helix protein engineered to bind to EGFR with low nanomolar affinity (41). The untargeted counterpart was Affibody negative control, which maintains the same three-helix structure and size as the anti-EGFR agent and shows no binding affinity to EGFR.…”

Section: Resultsmentioning

confidence: 99%

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo

Davis

Samkoe²,

Tichauer

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies.

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Section: Resultsmentioning

confidence: 99%

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo

Davis

Samkoe²,

Tichauer

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…The bs affibody molecule was injected at different concentrations over the flow-cell surfaces of a sensor chip containing immobilized HER2 and EGFR respectively. The affinities (equilibrium dissociation constant, K D ) of the monomeric affibody molecules were previously determined to be 22 pM for Z HER2:342 [30] and 5.4 nM for Z EGFR:1907 [31]. The dimer format of each affibody molecule in the bs affibody protein will most likely give an additional increase in functional affinity (avidity), as demonstrated previously [38,39].…”

Section: Single and Dual Binding Biosensor Analysismentioning

confidence: 59%

“…Affibody molecules have shown potential in a wide variety of applications, e.g. as detection reagents [23][24][25], in bioseparation [26][27][28], for depletion in proteomics applications [29] and for tumourtargeting applications [30][31][32][33][34]. The small size (∼ 6.5 kDa), generally high stability, efficient production in Eschercihia coli or by chemical synthesis and straightforward thiol-based site-specific coupling or labelling are favourable properties for affinity proteins [35].…”

Section: Introductionmentioning

confidence: 99%

Engineering and characterization of a bispecific HER2 × EGFR‐binding affibody molecule

Friedman

Lindström

Ekerljung

et al. 2009

Biotech and App Biochem

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“…We used SpyLigase to polymerize affibodies to create multivalent chains for sensitive cell capture. Affibodies are a nonimmunoglobulin scaffold, consisting of a three-helix bundle, selected for high-affinity binding through phage display, and easily expressed in E. coli (21). The initial affibody we modified bound strongly and specifically to epidermal growth factor receptor (EGFR) (21), a tyrosine kinase expressed on many cancer cells and a target antigen in CTC isolation (22,23).…”

Section: Resultsmentioning

confidence: 99%

SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

Fierer

Veggiani

Howarth

2014

Proc. Natl. Acad. Sci. U.S.A.

155

162

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Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein-peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide-peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide-peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture.bionanotechnology | synthetic biology | metastasis | antibody | nanobiotechnology

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Directed Evolution to Low Nanomolar Affinity of a Tumor-Targeting Epidermal Growth Factor Receptor-Binding Affibody Molecule

Cited by 139 publications

References 51 publications

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo

Engineering and characterization of a bispecific HER2 × EGFR‐binding affibody molecule

SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

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