Joong-jae Lee

Design of a binding scaffold based on variable lymphocyte receptors of jawless vertebrates by module engineering

Repeat proteins have recently been of great interest as potential alternatives to immunoglobulin antibodies due to their unique structural and biophysical features. We here present the development of a binding scaffold based on variable lymphocyte receptors, which are nonimmunoglobulin antibodies composed of Leucine-rich repeat modules in jawless vertebrates, by module engineering. A template scaffold was first constructed by joining consensus repeat modules between the N- and C-capping motifs of variable lymphocyte receptors. The N-terminal domain of the template scaffold was redesigned based on the internalin-B cap by analyzing the modular similarity between the respective repeat units using a computational approach. The newly designed scaffold, termed “Repebody,” showed a high level of soluble expression in bacteria, displaying high thermodynamic and pH stabilities. Ease of molecular engineering was shown by designing repebodies specific for myeloid differentiation protein-2 and hen egg lysozyme, respectively, by a rational approach. The crystal structures of designed repebodies were determined to elucidate the structural features and interaction interfaces. We demonstrate general applicability of the scaffold by selecting repebodies with different binding affinities for interleukin-6 using phage display.

Protein binder for affinity purification of human immunoglobulin antibodies.

The importance of a downstream process for the purification of immunoglobulin antibodies is increasing with the growing application of monoclonal antibodies in many different areas. Although protein A is most commonly used for the affinity purification of antibodies, certain properties could be further improved: higher stability in alkaline solution and milder elution condition. Herein, we present the development of Fc-specific repebody by modular engineering approach and its potential as an affinity ligand for purification of human immunoglobulin antibodies. We previously developed the repebody scaffold composed of Leucine-rich repeat (LRR) modules. The scaffold was shown to be highly stable over a wide range of pH and temperature, exhibiting a modular architecture. We first selected a repebody that binds the Fc fragment of human immunoglobulin G (IgG) through a phage display and increased its binding affinity up to 1.9 × 10(-7) M in a module-by-module approach. The utility of the Fc-specific repebody was demonstrated by the performance of an immobilized repebody in affinity purification of antibodies from a mammalian cell-cultured medium. Bound-antibodies on an immobilized repebody were shown to be eluted at pH 4.0 with high purity (>94.6%) and recovery yield (>95.7%). The immobilized repebody allowed a repetitive purification process more than ten times without any loss of binding capability. The repebody remained almost intact even after incubation with 0.5 M NaOH for 15 days. The present approach could be effectively used for developing a repeat module-based binder for other target molecules for affinity purification.

Synthesis, electrical and optical properties of asymmetrically monoalkoxy-substituted PPV derivatives

Abstract Poly(2-methoxy-1,4-phenylenevinylene), PMPV, and its copolymers containing both unsubstituted and 2-methoxy-1,4-phenylenevinylene (MPV) units were prepared in thin films from their water-soluble proecursor polymers, and monobutoxy- and monododecyloxy-substituted PPV derivatives were also prepared via the water-soluble precursor method. Drawn films (L/Lo = 6) of PMPV could be doped with I2 vapor and FeCl3 to give conductivities of 3.2 and 4.0 Scm−1, respectively. Conductivity values of long alkoxy-substituted PPVs were decreased by increasing the carbon number of mono substituted alkoxy side chain. Conductivity of FeCl3 doped copolymer films ranged from 1–100 Scm−1 depending on the composition of the copolymers. The third-order nonlinear optical susceptibility, χ(3)(3ω; ω,ω,ω) for PMPV thin film has been investigated by degenerate four-wave mixing technique using 400 femtosecond pulses at 602nm. A relatively large 3rd-order susceptibilty value (χ(3) = 7.9 × 10−10 esμ) with a subpicosecond response was observed.

Tracking protein-protein interaction and localization in living cells using a high-affinity molecular binder.

Probing protein-protein interactions in living cells is crucial for understanding the protein functions and developing drugs. Small-sized protein binders are considered effective and useful for such analysis. Here we describe the development and use of a repebody, which is a protein binder composed of LRR (Leucine-rich repeat) modules, for tracking protein-protein interaction and localization in real-time through live-cell imaging. A repebody with high affinity for a red fluorescent protein was selected through a phage display, fused with a green fluorescent protein, and applied for tracing a red fluorescent protein-fused target protein in mammalian cells. The potential and utility of our approach was demonstrated by tracking the rapamycin-mediated interaction between FKBP12-rapamycin binding (FRB) domain and a FK506-binding protein (FKBP) and their localization by live-cell imaging. The present approach can be widely used for the analysis of protein-protein interaction and an understanding of complex biological processes in living cells.

Dissecting the critical factors for thermodynamic stability of modular proteins using molecular modeling approach.

Repeat proteins have recently attracted much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural and biophysical features. In particular, repeat proteins show high stability against temperature and chaotic agents. Despite many studies, structural features for the stability of repeat proteins remain poorly understood. Here we present an interesting result from in silico analyses pursuing the factors which affect the stability of repeat proteins. Previously developed repebody structure based on variable lymphocytes receptors (VLRs) which consists of leucine-rich repeat (LRR) modules was used as initial structure for the present study. We constructed extra six repebody structures with varying numbers of repeat modules and those structures were used for molecular dynamics simulations. For the structures, the intramolecular interactions including backbone H-bonds, van der Waals energy, and hydrophobicity were investigated and then the radius of gyration, solvent-accessible surface area, ratio of secondary structure, and hydration free energy were also calculated to find out the relationship between the number of LRR modules and stability of the protein. Our results show that the intramolecular interactions lead to more compact structure and smaller surface area of the repebodies, which are critical for the stability of repeat proteins. The other features were also well compatible with the experimental results. Based on our observations, the repebody-5 was proposed as the best structure from the all repebodies in structure optimization process. The present study successfully demonstrated that our computer-based molecular modeling approach can significantly contribute to the experiment-based protein engineering challenge.

A High-Affinity Repebody for Molecular Imaging of EGFR-Expressing Malignant Tumors

The accurate detection of disease-related biomarkers is crucial for the early diagnosis and management of disease in personalized medicine. Here, we present a molecular imaging of human epidermal growth factor receptor (EGFR)-expressing malignant tumors using an EGFR-specific repebody composed of leucine-rich repeat (LRR) modules. The repebody was labeled with either a fluorescent dye or radioisotope, and used for imaging of EGFR-expressing malignant tumors using an optical method and positron emission tomography. Our approach enabled visualization of the status of EGFR expression, allowing quantitative evaluation in whole tumors, which correlated well with the EGFR expression levels in mouse or patients-derived colon cancers. The present approach can be effectively used for the accurate detection of EGFR-expressing cancers, assisting in the development of a tool for detecting other disease biomarkers.

Soluble expression of human glycoprotein Ibα in Escherichia coli through replacement of the N-terminal capping domain.

Glycoprotein Ibα (GpIbα), a family of LRR (leucine-rich repeat) proteins, is a membrane protein on the platelet, and plays an important role in atherothrombotic events. The complex formation of GpIbα with the von Willebrand Factor (vWF) has been revealed to lead to acute coronary syndrome (ACS) or stroke. A considerable attention has been paid to understand the biological functions of GpIbα and its regulation. However, difficulty with the soluble expression of human GpIbα in bacteria has hampered the relevant research. Herein, we present a soluble expression of GpIbα in Escherichiacoli by replacing the N-terminal capping domain of GpIbα with that of Internalin B using a computational approach. The resulting protein was expressed as a soluble form in E. coli, maintaining its structural feature and binding property for vWF. The present approach can be broadly used for the soluble expression of human LRR proteins in E. coli.

64Cu-Labeled Repebody Molecules for Imaging of Epidermal Growth Factor Receptor–Expressing Tumors

The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptors and is overexpressed in many tumor types. A repebody is a newly designed nonantibody protein scaffold for tumor targeting that contains leucine-rich repeat modules. In this study, 3 64Cu-labeled anti-EGFR repebodies with different chelators were synthesized, and their biologic characteristics were assessed in cultured cells and tumor-bearing mice. Methods: Repebodies were synthesized with the chelators 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-N,N′,N,″-triacetic acid trihydrochloride ([p-SCN-Bn]-NOTA), 2,2′,2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-N-hydroxysuccinimide ester), or 1-(p-isothiocyanatobenzyl)diethylenetriamine pentaacetic acid trihydrochloride ([p-SCN-Bn]-DTPA) in 1.0 M NaHCO3 buffer (pH 9.2) for 24 h. Purified NOTA-, DOTA-, and DTPA-conjugated repebody were radiolabeled with 64Cu in 0.1 M NH4OAc buffer (pH 5.5). To compare the EGFR-binding affinities of the repebodies, cellular uptake studies were performed with the human non–small cell lung cancer cell line H1650 (high expression of EGFR) and the human colon adenocarcinoma cell line SW620 (low expression of EGFR). Biodistribution and small-animal PET imaging studies were performed using H1650 tumor–bearing mice. Results: Radiochemical yields of the 64Cu-labeled repebodies were approximately 70%–80%. Cellular uptake of the NOTA-, DOTA-, and DTPA-repebodies was over 4-fold higher in H1650 cells than in SW620 cells at 1 h. The 3 repebodies had accumulated specifically in H1650 tumor–bearing nude mice by 1 h after intravenous injection and were retained for over 24 h, as measured by the percentage injected dose per gram of tissue (%ID/g). Tumor uptake of all repebodies increased from 1 to 6 h (at 1 h, 6.28, 8.46, and 6.91 %ID/g for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 9.4, 8.28, and 10.1 %ID/g, respectively). H1650 tumors were clearly visible after injection of each repebody, with high tumor-to-background ratios (at 1 h, 3.43, 4.89, and 2.38 for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 3.05, 4.36, and 2.08; at 24 h, 3.81, 4.58, and 2.86). Conclusion: The 3 64Cu-repebody complexes demonstrated specific and rapid uptake in EGFR-expressing tumors within 1 h and may have potential as novel EGFR imaging agents for PET.

Radiolabeling and preliminary biodistribution study of 99mTc-labeled antibody-mimetic scaffold protein repebody for initial clearance properties

Antibody-mimetic proteins are intensively being developed for biomedical applications including tumor imaging and therapy. Among them, repebody is a new class of protein that consists of highly diverse leucine-rich repeat (LRR) modules. Although all possible biomedical applications with repebody are ongoing, its in vivo biodistribution and excretion pathway has not yet been explored. In this study, hexahistidine (His6)-tag bearing repebody (rEgH9) was labeled with [99mTc]-tricarbonyl, and biodistribution was performed following intravenous (I.V.) or intraperitoneal (I.P.) injection. Repebody protein was radiolabeled with high radiolabeling efficiency (>90%) and radiolabeled compound was more than 99% pure after purification. Biodistribution data indicates radiotracer has a rapid clearance from blood and excreted through the kidneys for intravenous (I.V.) injection, but comparatively slow clearance for an intraperitoneal (I.P.) injection. SPECT-CT images were found to be in agreement with biodistribution data, high activity was found inside kidneys. The observed result for rapid blood clearance and renal excretion of repebody (rEgH9) provide useful information for the further development of therapeutic strategy.

A dimeric form of a small-sized protein binder exhibits enhanced anti-tumor activity through prolonged blood circulation

ABSTRACT Small‐sized non‐antibody scaffolds have attracted considerable interest as alternatives to immunoglobulin antibodies. However, their short half‐life is considered a drawback in the development of therapeutic agents. Here we demonstrate that a homo‐dimeric form of a repebody enhances the anti‐tumor activity than a monomeric form through prolonged blood circulation. Spytag and spycatcher were genetically fused to the C‐terminus of a respective human IL‐6‐specific repebody, and the resulting two repebody constructs were mixed at an equimolar ratio to produce a homo‐dimeric form through interaction between spytag and spycatcher. The homo‐dimeric repebody was detected as a single band in the SDS‐PAGE analysis with an expected molecular size (78kDa), showing high stability and homogeneity. The dimeric repebody was shown to simultaneously accommodate two hIL‐6 molecules, and its binding affinity for hIL‐6 was estimated to be comparable to a monomeric repebody. The serum concentration of the dimeric repebody was observed to be about 5.5 times higher than a monomeric repebody, consequently leading to considerably higher tumor suppression effect in human tumor xenograft mice. The present approach can be effectively used for prolonging the blood half‐life of small‐sized protein binders, resulting in enhanced therapeutic efficacy.