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ez-ADiCon: A novel glyco-remodeling based strategy that enables preparation of homogenous antibody-drug conjugates via one-step enzymatic transglycosylation with payload-preloaded bi-antennary glycan complexes

Article

作者: Kanamori, Satoshi ; Chon, Hyongi ; Narumi, Hideki ; Nagahara, Takashi ; Ohara, Keiichiro ; Suzuki, Tomohiko ; Hibino, Kazuhiro

The conserved N-linked glycan at the Fc domain of recombinant monoclonal antibodies is an attractive target for site-specific payload conjugation for preparation of homogenous antibody-drug conjugates (ADCs). Here, we report a novel ADC constructing strategy, named "ez-ADiCon", that is achieved by one-step enzymatic transglycosylation of a payload-preloaded bi-antennary glycan oxazoline onto a deglycosylated antibody. In this method, a mixture of different glycoforms of the Fc-glycan is replaced with a pre-defined payload-linked glycan. Since two payloads are linked on each donor glycan substrate, efficient conjugation results in a highly homogenous ADC with mostly-four drug molecules per antibody, facilitating hydrophobic interaction chromatography analysis and purification. We validated this conjugation strategy using Monomethyl auristatin E (MMAE) and an anti-Human epidermal growth factor receptor 2 (anti-Her2) antibody as the model ADC components and demonstrated its target-specific in vitro cytotoxicity. Our novel conjugation strategy, ez-ADiCon, provides a new approach for the preparation of next generation ADCs.

2020-12-01·Bioscience, Biotechnology, and Biochemistry4区 · 工程技术

Crystal structure of a glycoside hydrolase family 68 β-fructosyltransferase from Beijerinckia indica subsp. indica in complex with fructose

4区 · 工程技术

Article

作者: Kitamura, Junichi ; Hirano, Katsuaki ; Nagaya, Mika ; Tamura, Keisuke ; Tonozuka, Takashi ; Kawai, Reika ; Tochio, Takumi ; Nishikawa, Atsushi ; Fujii, Tadashi

AbstractAn enzyme belonging to glycoside hydrolase family 68 (GH68) from Beijerinckia indica subsp. indica NBRC 3744 was expressed in Escherichia coli. Biochemical characterization showed that the enzyme was identified to be a β-fructosyltransferase (BiBftA). Crystallization of a full-length BiBftA was initially attempted, but no crystals were obtained. We constructed a variant in which 5 residues (Pro199-Gly203) and 13 residues (Leu522-Gln534) in potentially flexible regions were deleted, and we successfully crystallized this variant BiBftA. BiBftA is composed of a five-bladed β-propeller fold as in other GH68 enzymes. The structure of BiBftA in complex with fructose unexpectedly indicated that one β-fructofuranose (β-Fruf) molecule and one β-fructopyranose molecule bind to the catalytic pocket. The orientation of β-Fruf at subsite −1 is tilted from the orientation observed in most GH68 enzymes, presenting a second structure of a GH68 enzyme in complex with the tilted binding mode of β-Fruf.

2020-08-01·The Journal of Antibiotics4区 · 医学

Serine catabolism produces ROS, sensitizes cells to actin dysfunction, and suppresses cell growth in fission yeast

4区 · 医学

Article

作者: Tabuchi, Toshitsugu ; Kato, Taira ; Kakeya, Hideaki ; Kanou, Akihiko ; Yoshida, Minoru ; Nishimura, Shinichi ; Matsuyama, Akihisa

Serine is an essential component in organisms as a building block of biomolecules, a precursor of metabolites, an allosteric regulator of an enzyme, etc. This amino acid is thought to be a key metabolite in human diseases including cancers and infectious diseases. To understand the consequence of serine catabolism, we screened natural products to identify a fungal metabolite chaetoglobosin D (ChD) as a specific inhibitor of fission yeast cell growth when cultivated with serine as a sole nitrogen source. ChD targets actin, and actin mutant cells showed severe growth defect on serine medium. ROS accumulated in cells when cultivated in serine medium, while actin mutant cells showed increased sensitivity to oxidative stress. ROS production is a new aspect of serine metabolism, which might be involved in disease progression, and actin could be the drug target for curing serine-dependent symptoms.

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