Biomedical subjects

Kouhei Tsumoto

Publications and source records attributed to Kouhei Tsumoto.

59 records · Page 4Linked to original sources

PubMed2002

A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 in targeted cancer immunotherapy for bile duct carcinoma.

In cancer immunotherapy research, many bispecific antibodies (BsAbs) have been developed for directing T cells toward tumor cells. Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems. Therefore, recombinant BsAbs, termed diabodies, have attracted particular attention. We have previously produced an anti-MUC1 x anti-CD3 diabody (Mx3 diabody) in an Escherichia coli ( E. coli) expression system. In order to reinforce the antitumor effects of the Mx3 diabody, mutated superantigen staphylococcal enterotoxin A (SEA) D227A was genetically fused to the Mx3 diabody. The SEA D227A fusion Mx3 diabody (SEA D227A-Mx3 diabody) thus constructed showed remarkable MUC1-specific antitumor effects when used with effector cells (lymphokine-activated killer cells with T-cell phenotype [T-LAK] and peripheral blood mononuclear cells [PBMCs]). In the bile duct carcinoma (BDC)-xenografted severe combined immunodeficient (SCID) mouse model, it also demonstrated strong antitumor activity when administered i.v. together with T-LAK cells and interleukin-2 (IL-2). In this experiment, the complete disappearance of tumors was observed in 3 out of 6 mice, and the other 3 showed marked retardation of tumor growth. Therefore, the SEA D227A-Mx3 diabody is considered to be a promising reagent in specific targeted immunotherapy for BDC and other MUC1-positive carcinomas. This is the first report on a diabody that is effective in treating human solid cancers in the xenografted SCID mouse experimental model.

Animals↗

PubMed2002

Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera.

In mammalian cell culture, the selection of high producers is a critical step in efficient recombinant protein production. Drug-resistance selection has been commonly used, but does not always give a pure population of high producers. In this study, we propose a novel selection method in which the growth of high producers is specifically promoted. Two plasmids encoding (i) a hybrid receptor composed of the V(H) portion of anti-hen egg lysozyme antibody HyHEL-10 and an N-terminally truncated erythropoietin receptor (V(H)-EpoR), and (ii) a V(L)-EpoR fusion derived from the same construct as in (i), were employed. The second plasmid contained enhanced green fluorescent protein (EGFP) as a model recombinant protein that was flanked by the internal ribosomal entry sequence. Both plasmids were used simultaneously to transfect an IL-3-dependent murine myeloid cell line, 32D. The transfectants, after antigen selection in the absence of IL-3, showed a clear antigen-induced dose-dependent proliferation. In addition, a high EGFP expression level was observed by flow cytometry in comparison with the cells before antigen selection. The results clearly demonstrate the advantage of our method over conventional drug-resistance selection. We propose the term AMEGA (Antigen MEdiated Genetically-modified cell Amplification) for such an approach.

Journal Article↗

PubMed2002

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment.

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).

Animals↗

PubMedDate not supplied

Kinetics of ultrasonic disinfection of Escherichia coli in the presence of titanium dioxide particles.

The ultrasonic disinfection of Escherichia coli was carried out in the presence of anatase-type TiO2 particles, and the effectiveness of the combination of ultrasonic irradiation with TiO2 addition was verified. The rate constant was determined from the plot of the common logarithm of the survival cell ratio versus the ultrasonic irradiation time using first-order kinetics. In the absence of particles, the rate constant was proportional to the ultrasonic power. When ultrasonic disinfection was carried out in the presence of TiO2 particles, which have a radical generation ability, the rate of disinfection was remarkably faster than that in the absence of TiO2. In the presence of silica particles, which have no radical generation ability, the rate of disinfection was the same as that in the absence of TiO2. These results suggest that the radical generation ability of TiO2 appeared as a result of the ultrasonic irradiation. The effect of the amount of TiO2 on the rate of disinfection was also examined. The rate constant for disinfection in the presence of TiO2 was saturated to a certain value and was represented using the Langmuir-type equation. The proposed model well describes the effects of the ultrasonic power and the amount of TiO2 on the rate constant for disinfection.

Cell Survival↗

PubMedDate not supplied

Role of arginine in protein refolding, solubilization, and purification.

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.

Arginine↗