Izumi Kumagai

Tohoku University, Sendai-shi, Miyagi, Japan

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Publications (283)888.31 Total impact

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    ABSTRACT: Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.
    Nanoscale 11/2015; DOI:10.1039/C5NR06471F · 7.39 Impact Factor
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    ABSTRACT: We report the observation of kinesin driven quantum dots (QDs) trapped in a microtubule loop, allowing the investigation of moving QDs for a long time and an unprecedented long distance. The QD conjugates did not depart from our observational field of view, enabling the tracking of specific conjugates for more than 5 minutes. The unusually long run length and the periodicity caused by the loop track allow comparing and studying the trajectory of the kinesin driven QDs for more than 2 full laps, i.e. about 70 µm, enabling a statistical analysis of interactions of the same kinesin driven object with the same obstacle. The trajectories were extracted and analyzed from kymographs with a newly developed algorithm. Despite dispersion, several repetitive trajectory patterns can be identified. A method evaluating the similarity is introduced allowing a quantitative comparison between the trajectories. The velocity variations appear strongly correlated to the presence of obstacles. We discuss the reasons making this long continuous travel distances on the loop track possible.
    ACS Nano 10/2015; DOI:10.1021/acsnano.5b04348 · 12.88 Impact Factor
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    ABSTRACT: In nanotechnological devices, mass transport can be initiated by pressure driven flow, diffusion or by employing molecular motors. As the scale decreases, molecular motors can be helpful as they are not limited by increased viscous resistance. Moreover, molecular motors can move against diffusion gradients and are naturally fitted for nanoscale transportation. Among motor proteins, kinesin has particular potential for lab-on-a-chip applications. It can be used for sorting, concentrating or as a mechanical sensor. When bound to a surface, kinesin motors propel microtubules in random directions, depending on their landing orientation. In order to circumvent this complication, the microtubule motion should be confined or guided. To this end, dielectrophoretically aligned multi-walled-carbon nanotubes (MWCNT) can be employed as nanotracks. In order to control more precisely the spatial repartition of the MWCNTs, a screening method has been implemented and tested. Polygonal patterns have been fabricated with the aim of studying the guiding and the microtubule displacement between MWCNT segments. Microtubules are observed to transfer between MWCNT segments, a prerequisite for the guiding of microtubules in MWCNT circuit-based biodevices. The effect of the MWCNT organization (crenellated or hexagonal) on the MT travel distance has been investigated as well.
    Biomedical Microdevices 08/2015; 17(4):9978. DOI:10.1007/s10544-015-9978-1 · 2.88 Impact Factor
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    Ryutaro Asano · Izumi Kumagai ·
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    ABSTRACT: Although antibodies have been used as molecularly targeted agents for difficult-to-treat diseases such as cancers, the high production costs associated with mammalian expression systems continue to be a drawback. In addition, the clinical efficacy of conventional IgG antibodies is limited. Several types of recombinant antibody (e.g. fused with anticancer drugs, multivalent, or multispecific) have been designed in efforts to develop next-generation antibodies with higher functionality. We used protein engineering to construct several anticancer recombinant antibodies by developing bispecific antibodies that induced specific antitumor effects against cancer cells through the recruitment of lymphocytes. We found that a humanized small bispecific antibody (Ex3) that targets epidermal growth factor receptor on tumor cells and CD3 on T lymphocytes had marked anticancer activity. Furthermore, the function of Ex3 was enhanced by fusion with the human Fc region, domain rearrangement, multimerization, and affinity maturation; a combination of these modifications showed at least additive cytotoxic effects. Interestingly, merely rearranging the domain order of Ex3 induced substantial cytotoxic enhancements, even though the structural format remained the same. Here, we describe our efforts to develop highly functional bispecific antibodies as next-generation therapeutic antibodies using protein engineering.
    YAKUGAKU ZASSHI 07/2015; 135(7):851-6. DOI:10.1248/yakushi.15-00007-2 · 0.26 Impact Factor
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    ABSTRACT: The interaction between biomolecules and inorganic materials has gained much interest for the development of sensors and highly controlled materials in scale and composition.1 For this purpose, 4F2, a VHH antibody with high affinity and specificity toward zinc oxide (ZnO) surfaces has been constructed.2 Here, we report that 4F2 also has an affinity toward zinc ion (Zn2+). The stability of 4F2 and its Zn2+ complexes was determined using differential scanning calorimetry. We then determined the binding affinity between 4F2 and Zn2+ by using isothermal calorimetry. To further elucidate the molecular binding mechanism of 4F2 toward Zn2+, we applied the 1H-15N heteronuclear single quantum coherence NMR technique. We found that 4F2 recognized Zn2+ near the complementarity determining region 3 (CDR3). Our results provide the basis for the recognition mechanism of ZnO by 4F2.
    Chemistry Letters 01/2015; 44(10). DOI:10.1246/cl.150537 · 1.23 Impact Factor
  • Ryutaro Asano · Izumi Kumagai ·

    Seikagaku. The Journal of Japanese Biochemical Society 08/2014; 86(4):469-73. · 0.04 Impact Factor
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    ABSTRACT: One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)-variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH-VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.
    mAbs 07/2014; 6(5). DOI:10.4161/mabs.29445 · 4.56 Impact Factor
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    ABSTRACT: Gliding of microtubule filaments on surfaces coated with the motor protein kinesin has potential applications for nano-scale devices. The ability to guide the gliding direction in three dimensions allows the fabrication of tracks of arbitrary geometry in space. Here, we achieve this by using kinesin-coated glass wires of micrometer diameter range. Unlike previous methods in which the guiding tracks are fixed on flat two-dimensional surfaces, the flexibility of glass wires in shape and size facilitates building in-vitro devices that have deformable tracks.
    Biomedical Microdevices 04/2014; 16(4). DOI:10.1007/s10544-014-9852-6 · 2.88 Impact Factor
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    ABSTRACT: As a complementary tool to nanofluidics, biomolecular based transport is envisioned for nanotechnological devices. We report a new method for guiding microtubule shuttles on multi-walled carbon nanotube tracks, aligned by dielectrophoresis on a functionalized surface. In the absence of electric field and in fluid flow, alignment is maintained. The directed translocation of kinesin propelled microtubules has been investigated using fluorescence microscopy. To our knowledge, this is the first demonstration of microtubules gliding along carbon nanotubes.
    Nano Letters 02/2014; 14(2). DOI:10.1021/nl4042388 · 13.59 Impact Factor
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    ABSTRACT: N-Glycosylation of therapeutic antibodies contributes not only to their biological function, but also to their stability and tendency to aggregate. Here, we investigated the impact of the glycosylation status of an aggregated antibody that accumulated during the bioreactor culture of Chinese hamster ovary cells. High-performance liquid chromatography analysis showed that there was no apparent difference in the glycosylation patterns of monomeric, dimeric, and large aggregated forms of the antibody. In contrast, lectin binding assays, which enable the total amounts of specific sugar residues to be detected, showed that both galactose and fucose residues in dimers and large aggregates were reduced to 70-80% of the amount in monomers. These results strongly suggest that the lack of N-linked oligosaccharides, a result of deglycosylation or aglycosylation, occurred in a proportion of the dimeric and large aggregated components. The present study demonstrates that glycosylation heterogeneities are a potential cause of antibody aggregation in cell culture of Chinese hamster ovary cells, and that the lack of N-glycosylation promotes the formation of dimers and finally results in large aggregates.
    Journal of Bioscience and Bioengineering 12/2013; 117(5). DOI:10.1016/j.jbiosc.2013.11.001 · 1.88 Impact Factor
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    ABSTRACT: The aggregation of therapeutic antibodies during the manufacturing process is problematic because of the potential risks posed by the aggregates, such as an unexpected immune response. One of the hallmark effects of trehalose, a disaccharide consisting of two alpha-glucose units, is as a chemical chaperone with anti-aggregation activity. In this study, Chinese hamster ovary (CHO) cell line producing a diabody-type bispecific antibody were cultured in medium containing trehalose and the aggregation of the secreted proteins during the culture process was analyzed. An analysis of the various forms of the antibody (monomeric, dimeric, and large aggregates) showed that trehalose decreased the relative content of large aggregates by two thirds. The aggregation kinetics indicated that trehalose directly inhibited the polymerization and aggregation steps in a nucleation-dependent aggregation mechanism. Moreover, both specific and volumetric antibody production were increased in CHO cells cultured in trehalose-containing medium. Thus, the addition of trehalose to recombinant CHO cell cultures would offer a practical strategy for quality improvement in the production of therapeutic antibodies.
    Journal of Bioscience and Bioengineering 12/2013; 117(5). DOI:10.1016/j.jbiosc.2013.10.022 · 1.88 Impact Factor
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    ABSTRACT: The grating substrate covered with metal layer, plasmonic chip, and the bispecific antibody can make a key role for sensitive detection of a marker protein with immunosensor, because of the provision of enhanced fluorescence signal and the preparation of sensor surface densely modified with capture antibody, respectively. In this study, one of tumor markers, soluble epidermal growth factor receptor (s-EGFR), was selected as target to be detected. The ZnO and Ag-coated plasmonic chip with the precise regularity and appropriate duty ratio in the periodic structure further enhanced the fluorescence intensity. As for the sensor surface modification with capture antibody, bispecific antibody (anti-sEGFR x anti-ZnO antibody), the concentrated bispecific antibody solution was found to nonlinearly form a surface densely immobilized with antibody, because the binding process of bispecific antibody to ZnO surface can be competitive process with adsorption of phosphate. As a result, the interface on the plasmonic chip provided the 300 x enhanced fluorescence signal compared with that on a ZnO-coated glass slide, and therefore, s-EGFR was found to be quantitatively detected in a wide concentration range from 10 nM to 700 fM on our plasmonic surface.
    ACS Applied Materials & Interfaces 08/2013; 5(17). DOI:10.1021/am402173y · 6.72 Impact Factor
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    ABSTRACT: The construction of antibody fragments has the potential to reduce the high cost of therapeutic antibody production, but the structures of these fragments, with monovalency and the lack of an Fc region, can lead to reduced function. Multimerization is one strategy for recovering function that also yields better tumor-to-blood ratios than IgGs or monomeric antibody fragments because of rapid tumor uptake and clearance. Here, we constructed single-chain variable fragment (scFv) multimers by modifying the linker length and domain order of the humanized anti-EGFR antibody 528 (h528) and tested their ability to inhibit tumor growth. The h528 scFv multimers, expressed using a bacterial expression system, were successfully fractionated and inhibited cancer growth in a multimerization-dependent manner, whereas the h528 scFv monomer showed no inhibition. h528 scFv trimers with the VH-VL domain order and no linkers showed the highest in vitro and in vivo anti-tumor effects, which were comparable to those of the approved anti-EGFR therapeutic antibodies Cetuximab and Panitumumab. The trimers were also structurally stable in vitro and in vivo, which may be attributable to a strong interaction between the VH and VL of h528 Fv. Thus, h528 scFv multimers, especially trimers, are attractive as the next generation of anti-EGFR therapeutic antibodies and offer the possibility of low cost production. This article is protected by copyright. All rights reserved.
    FEBS Journal 07/2013; 280(19). DOI:10.1111/febs.12451 · 4.00 Impact Factor
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    ABSTRACT: To improve the efficiency of conventional gene amplification systems, the effect of cell cycle modification during the gene amplification process on IgG production was investigated in Chinese hamster ovary (CHO) cells. The full-length cDNA of CHO cell division cycle 25 homolog A (Cdc25A) was introduced into CHO DG44 cells and the effects of CDC25A overexpression on the cell cycle, transgene copy number and IgG productivity were examined. Both wild-type and mutated CDC25A-overexpressing CHO cells showed a rapid increase in transgene copy number compared with mock cells during the gene amplification process, in both cell pools and individual clones. High-producing clones were obtained with high frequency in CDC25A-overexpressing cell pools. The specific production rate of the isolated clone CHO SD-S23 was up to 2.9-fold higher than that of mock cells in the presence of 250 nM methotrexate (MTX). Cell cycle analysis revealed that the G2 to M phase transition rate was increased ∼1.5-fold in CDC25A-overexpressing CHO cells under MTX treatment. Our results show the improvement of conventional gene amplification systems via cell cycle engineering at an early stage of cell line development.
    Journal of Bioscience and Bioengineering 06/2013; 116(6). DOI:10.1016/j.jbiosc.2013.05.032 · 1.88 Impact Factor
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    ABSTRACT: Plasmonic chips, which are grating replicas coated with thin metal layers and overlayers such as ZnO, were applied in immunosensors to improve their detection sensitivity. Fluorescence from labeled antibodies bound to plasmonic chips can be enhanced on the basis of a grating-coupled surface plasmon resonance (GC-SPR) field. In this study, as one of the representative candidate protein markers for brain disorders, the brain-derived neurotrophic factor (BDNF) was quantitatively measured by sandwich assay on a plasmonic chip and detected on our plasmonic chip in the concentration of 5-7 ng/mL within 40 min. Furthermore, BDNF was detected in the blood sera from three types of mice: wild-type mice and two types of mutant mice. This technique is promising as a new clinical diagnosis tool for brain disorders based on scientific evidence such as blood test results. (C) 2013 The Japan Society of Applied Physics
    Japanese Journal of Applied Physics 06/2013; 52(6S):06GK01. DOI:10.7567/JJAP.52.06GK01 · 1.13 Impact Factor
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    ABSTRACT: Cellulosomes, which are assemblies of cellulases with various catalytic functions on a giant scaffoldin protein with a carbohydrate-binding module (CBM), efficiently degrade solid cellulosic biomass by means of synergistically coupled hydrolysis reactions. In this study, we constructed hybrid nanocellulosomes from the biotinylated catalytic domains (CDs) of two catalytically divergent cellulases (an endoglucanase and a processive endoglucanase) and biotinylated CBMs by clustering the domains and modules on streptavidin-conjugated nanoparticles. Nanocellulosomes constructed by separately clustering each type of CD with multiple CBMs on nanoparticles showed 5-fold enhancement in cellulase degradation activity relative to that of the corresponding free CDs, and mixtures of the two types of nanocellulosomes gradually and synergistically enhanced cellulase degradation activity as the CBM valency increased (finally, 2.5 times). Clustering the two types of CD together on the same nanoparticle resulted in a greater synergistic effect that was independent of CBM valency; consequently, nanocellulosomes composed of equal amounts of the endo and endoprocessive CDs clustered on a nanoparticle along with multiple CBMs (CD/CBM = 7:23) showed the best cellulose degradation activity, producing 6.5 and 2.4 times the amount of reducing sugars produced from amorphous and crystalline cellulose, respectively, by the native free CDs and CBMs in the same proportions. Our results demonstrate that hybrid nanocellulosomes constructed from the building blocks of cellulases and cellulosomes modules have the potential to serve as high-performance artificial cellulosomes.
    ACS Catalysis 05/2013; 3(6):1342–1348. DOI:10.1021/cs400012v · 9.31 Impact Factor
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Publication Stats

5k Citations
888.31 Total Impact Points


  • 1996-2015
    • Tohoku University
      • • Department of Biomolecular Engineering
      • • Graduate School of Engineering
      Sendai-shi, Miyagi, Japan
  • 2009
    • University of Hyogo
      • Graduate School of Material Science
      Kōbe, Hyōgo, Japan
  • 1987-2002
    • The University of Tokyo
      • • Department of Chemistry and Biotechnology
      • • Department of Chemistry
      • • Department of Physics
      • • Department of Biotechnology
      Tōkyō, Japan
  • 1999
    • Sapporo Medical University
      Sapporo, Hokkaidō, Japan
  • 1997
    • Nippon Medical School
      • Department of Biochemistry and Molecular Biology
      Tokyo, Tokyo-to, Japan
  • 1989-1995
    • Aoyama Gakuin University
      • College of Science and Engineering
      Tokyo, Tokyo-to, Japan
  • 1992-1993
    • Gakushuin University
      • Institute for Biomolecular Science
      Edo, Tōkyō, Japan
  • 1982-1983
    • Freie Universität Berlin
      • Institute of Chemistry and Biochemistry
      Berlín, Berlin, Germany