Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies

Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan^®) and anti-Her2/neu IgG1 trastuzumab (Herceptin^®). ADCC is triggered upon the binding of lymphocyte receptors (FcγRs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.     

Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation

Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.     

On-line sample extraction and enrichment of non-steroidal anti-inflammatory drugs by pre-column in capillary liquid chromatography mass spectrometry

A rapid and sensitive analytical method has been developed for the simultaneous determination of 16 non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma by capillary liquid chromatography (LC) and quadrupole mass spectrometry with electrospray ionization operated in the negative ion mode. The sample clean-up and enrichment on a pre-column were accomplished on-line to improve the sensitivity. This method greatly reduced sample preparation time and sample volume compared with off-line sample extraction methods and conventional LC methods, respectively. The recoveries of NSAIDs from human plasma were 56.7-96.9%. The total analytical time for a single analytical run was approximately 15 min. The detection limits of NSAIDs were 0.001-0.075 microg ml(-1) using a selected ion monitoring mode.     

Primary structural features of rosaceous S-RNases associated with gametophytic self-incompatibility

We isolated cDNA clones encoding five S-RNases (S_1-,_S3- , S_5-, S_6-, S_7-RNases) from pistils of Pyrus pyrifolia (Japanese pear), a member of the Rosaceae. Their amino acid sequences were aligned with those of other rosaceous S-RNases sequenced so far. A total of 76 conserved amino acid residues were stretched throughout the sequence, but were absent from the 51–66 region which was designated the hypervariable (HV) region. The phylogenetic tree of rosaceous S-RNases showed that S-RNase polymorphism predated the divergence of Pyrus and Malus. Pairwise comparison of these S-RNases detected two highly homologous pairs, P. pyrifolia S_1- and S_4-RNases (90.0%) and P. pyrifolia S_3- and S_5-RNases (95.5%). The positions of amino acid substitutions between S_1- and S_4-RNases were spread over the entire region, but in the pair of S_3- and S_5-RNases, amino acid substitutions were found in the 21–90 region including the HV region. The substitutions in this restricted region appear to be sufficient to discriminate between S₃ and S₅ pollen and to trigger the self-incompatible reaction.     

Application of microcolumn ion chromatography using anion exchangers modified with dextran sulfate for the determination of alkali and alkaline-earth metal ions

Microcolumn ion chromatography using anion exchangers modified with dextran sulfate has been applied to the determination of alkali and alkaline-earth metal ions contained in guinea pig serum and bovine serum. These serums contained Na⁺, NH₄⁺, K⁺, Mg²⁺ and Ca²⁺ and they were indirectly detected at 200 nm. The determination was done without any pretreatment procedure other than dilution.     

Multifunctional hydrolytic catalysis: VI. Catalytic hydrolysis of p-nitrophenyl acetate by N-(4-imidazolylmethyl)benzohydroxamic acid

A bifunctional catalyst, N-(4-imidazolylmethyl)benzohydroxamic acid, was synthesized from benzohydroxamic acid and chloromethylimidazole, and used for the hydrolysis of p-nitrophenyl acetate. The reaction proceeded via the formation of the acetyl hydroxamate and its subsequent decomposition. The deacylation step was shown to be general base-catalyzed by the intramolecular imidazole group on the basis of the deuterium solvent kinetic isotope effect of 2.0. The efficiency of water attack on the acetyl hydroxamate was enhanced 130-fold by the imidazole group. The catalytic process is compared with the reactions of related monofunctional compounds, and finally its significance as a model of the charge relay system is discussed.     

The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity

An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes alpha1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody.