Double knockdown of α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC

<p>Abstract</p> <p>Background</p> <p>Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans <it>in vivo</it>. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies.</p> <p>Results</p> <p>Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. α1,6-fucosyltransferase (<it>FUT8</it>), GDP-mannose 4,6-dehydratase (<it>GMD</it>), and GDP-fucose transporter (<it>GFT</it>), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of <it>FUT8 </it>and <it>GMD </it>siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with <it>GFT </it>siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of <it>FUT8 </it>and <it>GMD</it>, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture.</p> <p>Conclusion</p> <p>Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of <it>FUT8 </it>and <it>GMD </it>in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies.</p

トクシマシ シロヤマ ノ ホルトノキ ノ スイジャク コシ ノ ゲンイン ニツイテ : ホルトノキ イオウビョウ オ ヒキオコス ファイトプラズマ ノ シンコク ナ カンセン ジョウキョウ

Elaeocarpus sylvestris var. ellipticus was a dominant tree species in Mt. Shiroyama (castle mountain) in the Tokushima Central Park until 1970s, however, most of the E. sylvestris trees have died. In this study we investigated whether the death has been caused by Elaeocarpus yellows. Results of nested PCR revealed that DNA of phytoplasma, the pathogen of Elaeocarpus yellows, was found in all E. sylvestris trees in Mt. Shiroyama and about 80% E. sylvestris trees in the Tokushima Central Park. Results of PCR-RFLP showed that the all the trees had the same DNA type of phytoplasma, and it was identical to the one that had been found in Japan. These results indicated that the recent decrease of E. sylvestris in Mt. Shiroyama is due to Elaeocarpus yellows

Threonine 138 is crucial for the Quaternary Structure and the Thermostability of Thermus thermophilus Inorganic Pyrophosphatase.

Inorganic pyrophosphatase (EC. 3.6.1.1) from Thermus thermophilus (Tth PPase) forms the thermostable hexamer，and it was suggested from X-ray studies that its intersubunit interactions stabilize the whole molecule. However，the contribution of Thr138 at the intertrimer interface to quatemary structure and thermostability was unknown functionally. Therefore，we prepared four Thr138-substituted variants (T138A，V ，N ，and H) by site-directed mutagenesis. Then，thermostabilities of the enzyme activity and the quatemary structure for T138V and A were decreased relative to those of the wild type Tth PPase，whereas T138H and N variants remained much hexamer contents and the enzyme activity than T138V and A. Therefore，we suggest that the polar group in Thr138 of Tth PPase is more crucial than the methyl group for thermostability and quatemary structure，and it may contribute to the formation of stable trimer-trimer interface

トクシマシ シロヤマ ノ ホルトノキ ノ スイジャク コシ ノ ゲンイン ニツイテ : ホルトノキ イオウビョウ オ ヒキオコス ファイトプラズマ ノ シンコク ナ カンセン ジョウキョウ

Elaeocarpus sylvestris var. ellipticus was a dominant tree species in Mt. Shiroyama (castle mountain) in the Tokushima Central Park until 1970s, however, most of the E. sylvestris trees have died. In this study we investigated whether the death has been caused by Elaeocarpus yellows. Results of nested PCR revealed that DNA of phytoplasma, the pathogen of Elaeocarpus yellows, was found in all E. sylvestris trees in Mt. Shiroyama and about 80% E. sylvestris trees in the Tokushima Central Park. Results of PCR-RFLP showed that the all the trees had the same DNA type of phytoplasma, and it was identical to the one that had been found in Japan. These results indicated that the recent decrease of E. sylvestris in Mt. Shiroyama is due to Elaeocarpus yellows

Improved Adsorption of an Enterococcus faecalis Bacteriophage ΦEF24C with a Spontaneous Point Mutation

Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component

Case Report Severe Bradycardia Possibly due to a Local Anesthetic Oral Mucosal Injection during General Anesthesia

Local anesthesia may induce systemic complications leading to parasympathetic activity leading to bradycardia and hypotension. We report a case of a 50-year-old man undergoing dental surgery under general anesthesia who experienced severe bradycardia and hypotension after local anesthesia infiltration. Concerns regarding the utilization of a relatively large lumen injection needle for local anesthesia during general anesthesia are discussed

Thermostabilization by the Improvement of Intertrimeric Residues in Thermus thermophilus Inorganic Pyrophosphatase.

Inorganic pyrophosphatase (EC. 3.6.1.1) from Thermus thermophilus (Tth PPase)is a thermostable homohexamer of 174 amino acids，and its intertrimer interface is formed mainly by the symmetric α-helix A between subunits. Amino acids and their interactions composing intertrimer interface are different in hexameric Family I PPases，and then it was deduced that Tth PPase showed high thermostability because of stabilizing this interface by interactions of these residues. In this study，we focused on Thr138 and Ala141 residues in intertrimer interface of Tth PPase to confirm the relationship between intertrimeric residues and thermostability， and then improved their combination to His and Asp/Glu (HD or HE variant). As results，the HD variant showed the highest thermostability of enzyme activity，fluorescence spectra, and quaternary structure in the wild type Tth PPase and all variants. Especially，about 38% of hexamer and almost 40% of enzyme activity were observed in HD variant after heating even at 85℃. Therefore，we suggested that the conversion to a set of ionic His138 and Asp141 at intertrimer interface had increased the thermostability of Tth PPase，and then suppressed its thermal aggregation

Effect of Cys168 substitutions on the Thermostability and the Thermal Aggregation of Thermus thermophilus Inorganic Pyrophosphatase.

Thermus thermophilus Inorganic pyrophosphatase (Tth PPase) is comprised of homohexamer，and exhibits high thermostability. However，the thermal aggregation containing the cross-linked dimer was observed after heating above 85℃. Therefore，we focused on the sole cysteine (Cys168) in C-terminalregion，and evaluated the effects of substitutions at this position on thermostability and thermal aggregation of Tth PPase. Firstly，we prepared the four Cys168-substituted variants (C168A，L ，1，and F) by site-directed mutagenesis. Although all variants formed hexamer in native state，C168A variant exhibited the highest thermostabilities for the enzyme activity and quatemary structure in wild type and all variants，while the other variants decreased them drastically as the side chain at the 168 position was much more bulky and hydrophobic in Tth PPase. Moreover， suppression of thermal aggregation for C168A variant was observed in the ANS fluorescence experiments. Therefore，we suggest that the small volume and less hydrophobicity of side chain at 168 position may contribute to the conformational thermostability, and substitution with Ala is the most suitable for thermostabilization and suppression ofthermal aggregation of Tth PPase