Improvement of Artificial Antibody Secretion Using Supercharged Protein

Jiyeon Park(박지연),Heeju Choi(최희주),Hyejin Lee(이혜진),Jung Hoon Ahn(안정훈) 한국생명과학회 2020 생명과학회지 Vol.30 No.5

Repebody는 비면역 글로블린 인공 항체로 저렴하고 빠르게 생산 가능한 맞춤형 항체이다. 그러나 의료용 repebody의 생산은 저수율 및 복잡한 정제 공정으로 인해 여전히 어려움을 겪고 있다. Pseudomonas fluorescens의 ABC transporter를 사용한다면 생산 공정을 간소화하고 비용을 줄일 수는 있지만 repebody는 양전하를 띠어 분비 효율이 낮다. 따라서 등전점(pI)이 높은 repebody의 등전점을 낮추어 음전하를 띄도록 해야 한다. 이것을 위해 repebody의 N 말단과 C 말단에 연속된 아스파탐산을 붙여 보았지만 분비가 증가하지 않았다. 다른 방법으로 ABC transporter를 통한 repebody 분비 효율을 높이기 위해 repebody의 항원 결합 부위의 반대쪽에 존재하는 열다섯 개의 양전하 아미노산을 아스파탐산으로 변환하여 repebody 표면이 강한 음전하를 띠도록 하였다. 그 결과, 기존 repebody의 발현 단백질 당 분비효율은 21.2%였으나 변형한 과음전하 repebody의 분비효율은 58.5%로 향상되었다. 결론적으로 과음전하를 통해 만들어진 repebody는 P. fluorescens에 의해 세포 바깥에 분비 생산할 수 있었다. A repebody, an artificial non-immunoglobulin protein scaffold, is expected to be a solution in the search for faster, cheaper, and customizable antibodies. However, the production of medical repebodies remains difficult due to their low yield and the complex purification processes required. The Pseudomonas fluorescens ABC transporter system has been suggested as an efficient and cost-effective method for repebody production, but the total yield is low because of the secreted protein’s positive charge; thus, a repebody with a high isoelectric point needs to be changed into a more negatively charged protein for better secretion. To achieve this, we first attached oligo-aspartic acids to the N- and C-terminals of the repebody, but secretion efficiency was not enhanced significantly. Subsequently, we devised an alternative method for improved secretion efficiency by engineering fifteen positively charged amino acids to aspartic acid in the non-antigen binding sites of the repebody to give a high net negative charge. As a result, secretion efficiency was greatly enhanced from 21.2% (wildtype) to 58.5% (negatively supercharged). The negatively supercharged repebody was succussfully produced extracellularly by ABC transporter secretion system in P. fluorescens.

Genetically functionalized ferritin nanoparticles with a high-affinity protein binder for immunoassay and imaging

Kim, Jong-won,Heu, Woosung,Jeong, Sukyo,Kim, Hak-Sung Elsevier 2017 Analytica chimica acta Vol.988 No.-

<P><B>Abstract</B></P> <P>Molecular detection of target molecules with high sensitivity and specificity is of great significance in bio and medical sciences. Here, we present genetically functionalized ferritin nanoparticles with a high-affinity protein binder, and their utility as a signal generator in a variety of immunoassays and imaging. As a high-affinity protein binder, human IgG-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was used. The repebody was genetically fused to the N-terminal heavy-chain ferritin, and the resulting subunits were self-assembled to the repebody-ferritin nanoparticles composed of 24 subunits. The repebody-ferritin nanoparticles were shown to have a three-order of magnitude higher binding affinity toward human IgG than free repebody mainly owing to a decreased dissociation rate constant. The repebody-ferritin nanoparticles were conjugated with fluorescent dyes, and the resulting nanoparticles were used for western blotting, cell imaging, and flow cytometric analysis. The dye-labeled repebody-ferritin nanoparticles were shown to generate about 3-fold stronger fluorescent signals in immunoassays than monovalent repebody. The repebody-functionalized ferritin nanoparticles can be effectively used for sensitive and specific immunoassays and imaging in many areas.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Human IgG-specific repebody was genetically fused to N-terminus of ferritin subunit. </LI> <LI> The RbF-NPs exhibited 1000-fold higher binding affinity for human IgG than free repebody due to multivalency. </LI> <LI> The Cy3-laleled RbF-NPs generated stronger fluorescent signals than Cy3-labeled free repebody in immunoassays and imaging. </LI> <LI> The RbF-NPs can be effectively used in various types of immunoassays and imaging. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

Seo, Hyo-Deok,Lee, Joong-jae,Kim, Yu Jung,Hantschel, Oliver,Lee, Seung-Goo,Kim, Hak-Sung Elsevier 2017 Analytica chimica acta Vol.950 No.-

<P><B>Abstract</B></P> <P>Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL<SUP>−1</SUP> for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A human TNF-α (hTNF-α)-specific repebody was selected using a phage display. </LI> <LI> A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody. </LI> <LI> mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy. </LI> <LI> mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

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

Kim, Tae Yoon,Seo, Hyo-Deok,Lee, Joong-jae,Kang, Jung Ae,Kim, Woo Sik,Kim, Hye-Min,Song, Ha-Yeon,Park, Ji Min,Lee, Dong-Eun,Kim, Hak-Sung Elsevier 2018 Journal of controlled release Vol.279 No.-

<P><B>Abstract</B></P> <P>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 (78 kDa), 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.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Genetically engineered and self-assembled oncolytic protein nanoparticles for targeted cancer therapy

Lee, Joong-jae,Kang, Jung Ae,Ryu, Yiseul,Han, Sang-Soo,Nam, You Ree,Rho, Jong Kook,Choi, Dae Seong,Kang, Sun-Woong,Lee, Dong-Eun,Kim, Hak-Sung IPC Science and Technology Press 2017 Biomaterials Vol.120 No.-

<P><B>Abstract</B></P> <P>The integration of a targeted delivery with a tumour-selective agent has been considered an ideal platform for achieving high therapeutic efficacy and negligible side effects in cancer therapy. Here, we present engineered protein nanoparticles comprising a tumour-selective oncolytic protein and a targeting moiety as a new format for the targeted cancer therapy. Apoptin from chicken anaemia virus (CAV) was used as a tumour-selective apoptotic protein. An EGFR-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was employed to play a dual role as a tumour-targeting moiety and a fusion partner for producing apoptin nanoparticles in <I>E. coli</I>, respectively. The repebody was genetically fused to apoptin, and the resulting fusion protein was shown to self-assemble into supramolecular repebody-apoptin nanoparticles with high homogeneity and stability as a soluble form when expressed in <I>E. coli</I>. The repebody-apoptin nanoparticles showed a remarkable anti-tumour activity with negligible side effects in xenograft mice through a cooperative action of the two protein components with distinct functional roles. The repebody-apoptin nanoparticles can be developed as a systemic injectable and tumour-selective therapeutic protein for targeted cancer treatment.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

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

Pyo, Ayoung,Yun, Misun,Kim, Hyeon Sik,Kim, Tae-Yoon,Lee, Joong-jae,Kim, Jung Young,Lee, Sunwoo,Kwon, Seong Young,Bom, Hee-Seung,Kim, Hak-Sung,Kim, Dong-Yeon,Min, Jung-Joon Society of Nuclear Medicine 2018 The Journal of nuclear medicine Vol.59 No.2

<P>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 <SUP>64</SUP>Cu-labeled anti-EGFR repebodies with different chelators were synthesized, and their biologic characteristics were assessed in cultured cells and tumor-bearing mice. <B>Methods:</B> Repebodies were synthesized with the chelators 2-(<I>p</I>-isothiocyanatobenzyl)-1,4,7-triazacyclononane-<I>N,N′,N,″-</I>triacetic acid trihydrochloride ([<I>p</I>-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-<I>N</I>-hydroxysuccinimide ester), or 1-(<I>p</I>-isothiocyanatobenzyl)diethylenetriamine pentaacetic acid trihydrochloride ([<I>p</I>-SCN-Bn]-DTPA) in 1.0 M NaHCO<SUB>3</SUB> buffer (pH 9.2) for 24 h. Purified NOTA-, DOTA-, and DTPA-conjugated repebody were radiolabeled with <SUP>64</SUP>Cu in 0.1 M NH<SUB>4</SUB>OAc 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. <B>Results:</B> Radiochemical yields of the <SUP>64</SUP>Cu-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). <B>Conclusion:</B> The 3 <SUP>64</SUP>Cu-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.</P>

Effective suppression of C5a-induced proinflammatory response using anti-human C5a repebody

Hwang, D.E.,Choi, J.M.,Yang, C.S.,Lee, J.j.,Heu, W.,Jo, E.K.,Kim, H.S. Academic Press 2016 Biochemical and biophysical research communication Vol.477 No.4

The strongest anaphylatoxin, C5a, plays a critical role in the proinflammatory responses, causing the pathogenesis of a number of inflammatory diseases including sepsis, asthma, and rheumatoid arthritis. Inhibitors of C5a thus have great potential as therapeutics for various inflammatory disorders. Herein, we present the development of a high-affinity repebody against human C5a (hC5a), which effectively suppresses the proinflammatory response. A repebody scaffold composed of leucine-rich repeat (LRR) modules was previously developed as an alternative protein scaffold. A repebody specifically binding to hC5a was selected through a phage display, and its affinity was increased up to 5 nM using modular engineering. The repebody was shown to effectively inhibit the production of C5a-induced proinflammatory cytokines by human monocytes. To obtain insight into a mode of action by the repebody, we determined its crystal structure in complex with hC5a. A structural analysis revealed that the repebody binds to the D1 and D3 regions of hC5a, overlapping several epitope residues with the hC5a receptor (hC5aR). It is thus likely that the repebody suppresses the hC5a-mediated immune response in monocytes by blocking the binding of hC5a to its receptor. The anti-hC5a repebody can be developed as a potential therapeutic for C5a-involved inflammatory diseases.

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

Mushtaq, S.,Rho, J.K.,Kang, J.A.,Lee, J.J.,Kim, J.Y.,Nam, Y.R.,Yun, S.J.,Lee, G.H.,Park, S.H.,Lee, D.E.,Kim, H.S. Pergamon Press 2017 Bioorganic & medicinal chemistry letters Vol.27 No.22

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, it's in vivo biodistribution and excretion pathway has not yet been explored. In this study, hexahistidine (His<SUB>6</SUB>)-tag bearing repebody (rEgH9) was labeled with [<SUP>99m</SUP>Tc]-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.

Radiolabeling of antibody-mimetic scaffold protein with 99mTc tricarbonyl precursor via hexahistidine (His6)-tag

Ha Eun Shim,Dong-Eun Lee,Chang Heon Lee,Dae Seong Choi,Do Hee Kim 대한방사성의약품학회 2019 Journal of radiopharmaceuticals and molecular prob Vol.5 No.1

Recently, antibody-like scaffold proteins have received a great deal of interest in diagnosis and therapyapplications because of their intrinsic features that are often required for tumor imaging and therapy. Intrinsicissues that are associated with therapeutic application of antibody-like scaffold proteins, particularly in cancertreatment, include an efficient and straightforward radiolabeling for understanding in vivo biodistribution andexcretion route, and monitoring therapeutic responses. Herein, we report an efficient and straightforwardmethod for radiolabeling of antibody-like scaffold proteins with the [99mTc(OH2)3(CO)3]+ (99mTc-tricarbonyl) byusing a site-specific direct labeling method via hexahistidine-tag, which is a widely used for general purificationof recombinant proteins with His-affinity chromatography. Repebody is a new class of antibody-like scaffoldprotein that consists of highly diverse leucine-rich repeat (LRR) modules. Although all possible biomedicalapplications with repebody are ongoing, it’s in vivo biodistribution and excretion pathway has not yet beenexplored. In this study, hexahistidine (His6)-tag bearing repebody (rEgH9) was labeled with [99mTc]-tricarbonyl. Repebody protein was radiolabeled with high radiolabeling efficiency (>90%) and radiolabeled compound wasmore than 99% pure after purification. These results clearly demonstrate that the present radiolabeling methodwill be useful molecular imaging study.

Radiolabeling of antibody-mimetic scaffold protein with^99mTc tricarbonyl precursor via hexahistidine (His₆)-tag

Shim, Ha Eun,Kim, Do Hee,Lee, Chang Heon,Choi, Dae seong,Lee, Dong-Eun 대한방사성의약품학회 2019 Journal of radiopharmaceuticals and molecular prob Vol.5 No.1

Recently, antibody-like scaffold proteins have received a great deal of interest in diagnosis and therapy applications because of their intrinsic features that are often required for tumor imaging and therapy. Intrinsic issues that are associated with therapeutic application of antibody-like scaffold proteins, particularly in cancer treatment, include an efficient and straightforward radiolabeling for understanding in vivo biodistribution and excretion route, and monitoring therapeutic responses. Herein, we report an efficient and straightforward method for radiolabeling of antibody-like scaffold proteins with the $[^{99m}Tc(OH_2)_3(CO)_3]^+$ ($^{99m}Tc$-tricarbonyl) by using a site-specific direct labeling method via hexahistidine-tag, which is a widely used for general purification of recombinant proteins with His-affinity chromatography. Repebody is a new class of antibody-like scaffold protein that consists of highly diverse leucine-rich repeat (LRR) modules. Although all possible biomedical applications with repebody are ongoing, it's in vivo biodistribution and excretion pathway has not yet been explored. In this study, hexahistidine ($His_6$)-tag bearing repebody (rEgH9) was labeled with [$^{99m}Tc$]-tricarbonyl. Repebody protein was radiolabeled with high radiolabeling efficiency (>90%) and radiolabeled compound was more than 99% pure after purification. These results clearly demonstrate that the present radiolabeling method will be useful molecular imaging study.