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Engineered Charge Redistribution of Gp2 Proteins through Guided Diversity for Improved PET Imaging of Epidermal Growth Factor Receptor.

ABSTRACT: The Gp2 domain is a protein scaffold for synthetic ligand engineering. However, the native protein function results in a heterogeneous distribution of charge on the conserved surface, which may hinder further development and utility. We aim to modulate charge, without diminishing function, which is challenging in small proteins where each mutation is a significant fraction of protein structure. We constructed rationally guided combinatorial libraries with charge-neutralizing or charge-flipping mutations and sorted them, via yeast display and flow cytometry, for stability and target binding. Deep sequencing of functional variants revealed effective mutations both in clone-dependent contexts and broadly across binders to epidermal growth factor receptor (EGFR), insulin receptor, and immunoglobulin G. Functional mutants averaged 4.3 charge neutralizing mutations per domain while maintaining net negative charge. We evolved an EGFR-targeted Gp2 mutant that reduced charge density by 33%, maintained net charge, and improved charge distribution homogeneity while elevating thermal stability ( Tm = 87 ± 1 °C), improving binding specificity, and maintaining affinity ( Kd = 8.8 ± 0.6 nM). This molecule was conjugated with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid for 64Cu chelation and evaluated for physiological distribution in mice with xenografted A431 (EGFRhigh) and MDA-MB-435 (EGFRlow) tumors. Excised tissue gamma counting and positron emission tomography/computed tomography imaging revealed good EGFRhigh tumor signal (4.7 ± 0.5%ID/g) at 2 h post-injection and molecular specificity evidenced by low uptake in EGFRlow tumors (0.6 ± 0.1%ID/g, significantly lower than for non-charge-modified Gp2, p = 0.01). These results provide charge mutations for an improved Gp2 framework, validate an effective approach to charge engineering, and advance performance of physiological EGFR targeting for molecular imaging.

SUBMITTER: Case BA

PROVIDER: S-EPMC6051758 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Engineered Charge Redistribution of Gp2 Proteins through Guided Diversity for Improved PET Imaging of Epidermal Growth Factor Receptor.

Case Brett A BA Kruziki Max A MA Johnson Sadie M SM Hackel Benjamin J BJ

Bioconjugate chemistry 20180405 5

The Gp2 domain is a protein scaffold for synthetic ligand engineering. However, the native protein function results in a heterogeneous distribution of charge on the conserved surface, which may hinder further development and utility. We aim to modulate charge, without diminishing function, which is challenging in small proteins where each mutation is a significant fraction of protein structure. We constructed rationally guided combinatorial libraries with charge-neutralizing or charge-flipping m ...[more]

PMID: 29579383

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Project description:This purpose of this study is to determine the efficacy of a 45-amino acid Gp2 domain, engineered to bind to epidermal growth factor receptor (EGFR), as a positron emission tomography (PET) probe of EGFR in a xenograft mouse model. The EGFR-targeted Gp2 (Gp2-EGFR) and a nonbinding control were site-specifically labeled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Binding affinity was tested toward human EGFR and mouse EGFR. Biological activity on downstream EGFR signaling was examined in cell culture. DOTA-Gp2 molecules were labeled with 64Cu and intravenously injected (0.6-2.3 MBq) into mice bearing EGFRhigh (n = 7) and EGFRlow (n = 4) xenografted tumors. PET/computed tomography (CT) images were acquired at 45 min, 2 h, and 24 h. Dynamic PET (25 min) was also acquired. Tomography results were verified with gamma counting of resected tissues. Two-tailed t tests with unequal variances provided statistical comparison. DOTA-Gp2-EGFR bound strongly to human (KD = 7 ± 5 nM) and murine (KD = 29 ± 6 nM) EGFR, and nontargeted Gp2 had no detectable binding. Gp2-EGFR did not agonize EGFR nor antagonize EGF-EGFR. 64Cu-Gp2-EGFR tracer effectively localized to EGFRhigh tumors at 45 min (3.2 ± 0.5%ID/g). High specificity was observed with significantly lower uptake in EGFRlow tumors (0.9 ± 0.3%ID/g, p < 0.001), high tumor-to-background ratios (11 ± 6 tumor/muscle, p < 0.001). Nontargeted Gp2 tracer had low uptake in EGFRhigh tumors (0.5 ± 0.3%ID/g, p < 0.001). Similar data was observed at 2 h, and tumor signal was retained at 24 h (2.9 ± 0.3%ID/g). An engineered Gp2 PET imaging probe exhibited low background and target-specific EGFRhigh tumor uptake at 45 min, with tumor signal retained at 24 h postinjection, and compared favorably with published EGFR PET probes for alternative protein scaffolds. These beneficial in vivo characteristics, combined with thermal stability, efficient evolution, and small size of the Gp2 domain validate its use as a future class of molecular imaging agents.

Dataset Information

Engineered Charge Redistribution of Gp2 Proteins through Guided Diversity for Improved PET Imaging of Epidermal Growth Factor Receptor.

Publications

Engineered Charge Redistribution of Gp2 Proteins through Guided Diversity for Improved PET Imaging of Epidermal Growth Factor Receptor.

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