Biology subjects

Fujiyama, K.

Publications and source records attributed to Fujiyama, K..

3 recordsLinked to original sources

bioRxiv2022

Impact of glycoengineering and immunogenicity on the anti-cancer activity of a plant-made lectin-Fc fusion protein

Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core 1,3-fucose and {beta}1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFc{Delta}XF, has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumor mouse model, we found that systemic administration of AvFc{Delta}XF, but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumors, suggesting that Fc-mediated effector functions were integral. AvFc{Delta}XF treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of anti-drug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFc{Delta}XF before implanting tumors. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFc{Delta}XF was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFcs antitumor activity.

cancer biology↗

bioRxiv2021

Molecular basis for two stereoselective Diels-Alderases that produce decalin skeletons

Molecular chirality, discovered by Louis Pasteur in the middle of the 19th century1, is found in most primary and secondary metabolites. Particularly, the so-called natural products are rich in chiral centres2. The stereochemistry of natural products is strictly recognized in living organisms, and is thus closely related to their biological functions. The Diels-Alder (DA) reaction, which forms a six-membered ring with up to four chiral centres, is a fundamental practical reaction for C-C bond formation in synthetic chemistry3. Nature has also adopted this reaction to elaborate the complex structures of natural products using enzymes derived from various progenitor proteins4-7. Although enzymes catalysing the DA reaction, Diels-Alderases (DAases), have attracted increasing attention, little is known about the molecular mechanism by which they control the stereochemistry and perform catalysis. Here, we solved the X-ray crystal structures of a pair of decalin synthases, Fsa2 and Phm7, that catalyse intramolecular DA reactions to form enantiomeric decalin scaffolds during biosynthesis of the HIV-1 integrase inhibitor equisetin and its stereochemical opposite, phomasetin8,9. Based on the crystal structures, docking simulations followed by all-atom molecular dynamics simulations provided dynamic binding models demonstrating the folding of linear polyenoyl tetramic acid substrates in the binding pocket of these enzymes, explaining the stereoselectivity in the construction of decalin scaffolds. Site-directed mutagenesis studies verified the binding models and, in combination with density functional theory calculations, clarified how hydrophilic amino acid residues in the Phm7 pocket regulate and catalyse the stereoselective DA reaction. This study highlights the distinct molecular mechanisms of the enzymatic DA reaction and its stereoselectivity experimentally and computationally. We anticipate that clarified molecular mechanism herein provides not only the basic understanding how these important enzymes work but also the guiding principle to create artificial enzymes that produce designer bioactive molecules.

biochemistry↗

bioRxiv2019

Fully Human Monoclonal Antibodies Effectively Neutralizing Botulinum Neurotoxin Serotype B

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine-human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately [≥] 100 i.p. LD50/mg of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of M2 and M4 was able to completely neutralize BoNT/B1 with a potency greater than 10,000 i.p. LD50/mg of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication. IMPORTANCEBotulinum neurotoxins (BoNTs) produced by Clostridium botulinum and related species cause human botulism. Immunotherapy is the most effective treatment for botulism and equine immune serum formulations are used in cases of human botulism. However, these antisera may cause serum sickness or anaphylaxis. Additionally, the production of immune sera involves complicated and time-consuming manufacturing processes and quality management. Therefore, the development of safe, effective, and higher productive antibodies is required. Here we generated fully human monoclonal antibodies against serotype B BoNT (BoNT/B). We found that the combination of these antibodies (M2+M4) had potent and broad neutralization activity against BoNT/B, and showed therapeutic and preventive effects against botulism in mouse models. These data indicate that M2+M4 are promising candidates for the development of human therapeutics and prophylactics for BoNT/B intoxication.

microbiology↗