Mitsuo Satoh

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 α1,6-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.

Production of therapeutic antibodies with controlled fucosylation

ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; Fc, antibody constant region; FcγRIIIa, human Fcγ-receptor IIIa; IgG, immunoglobulin G; NK cell, natural killer cell; CHO, Chinese hamster ovary; EPO, erythropoietin; Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine; Gal, galactose; NANA; N-acetylneuraminic acid; FUT8, α-1,6 fucosyltransferase; GMD, GDP-mannose 4,6-dehydratase; FX, GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase; GFT, GDP-fucose transporter; siRNA, short interfering RNA; GnTIII, β-1,4-N-acetylglucosaminyltransferase; ManII, α-mannosidase II

Engineered therapeutic antibodies with improved effector functions

In the past decade, more than 20 therapeutic antibodies have been approved for clinical use and many others are now at the clinical and preclinical stage of development. Fragment crystallizable (Fc)‐dependent antibody functions, such as antibody‐dependent cell‐mediated cytotoxicity (ADCC), complement‐dependent cytotoxicity (CDC), and a long half‐life, have been suggested as important clinical mechanisms of therapeutic antibodies. These functions are primarily triggered through direct interaction of the Fc domain with its corresponding receptors: FcγRIIIa for ADCC, C1q for CDC, and neonatal Fc receptor for prolongation of the clearance rate. However, current antibody therapy still faces the critical issues of insufficient efficacy and the high cost of the therapeutic agents. A possible solution to these issues could be to engineer antibody molecules to enhance their antitumor activity, leading to improved therapeutic outcomes and reduced doses. Here, we review advanced Fc engineering approaches for the enhancement of effector functions, some of which are now ready for evaluation of their effectiveness in clinical trials. (Cancer Sci 2009; 100: 1566–1572)

A Peptidomics Strategy for Discovering Endogenous Bioactive Peptides

Peptide hormones and neuropeptides constitute an important class of naturally occurring peptides that are generated from precursor proteins by limited proteolytic processing. An important but unaddressed issue in peptidomics is to pin down novel bioactive peptides in a bulk of peptide sequences provided by tandem mass spectrometry. Here, we describe an approach to simultaneously screen for bioactive peptides and their target tissues. The principle behind this approach is to identify intact secretory peptides that have the ability to raise intracellular calcium levels. In practice, we used nanoflow liquid chromatography-tandem mass spectrometry to analyze peptides released by exocytosis from cultured cells. Peptide sequence information was utilized to deduce intact peptide forms, among which those highly conserved between species are selected and tested on an ex vivo calcium assay using tissue pieces from transgenic mice that systemically express the calcium indicator apoaequorin. The calcium assay can be applied to various cell types, including those not amenable to in vitro culture. We used this approach to identify novel bioactive neuropeptides derived from the neurosecretory protein VGF, which evoke a calcium response in the pituitary and hypothalamus.

Establishment of a novel monoclonal antibody against LGR5

LGR5 is an orphan G-protein-coupled receptor (GPCR) that is expressed on the cell surface membrane. LGR5 is reported to be overexpressed in colon, liver, and ovary tumor compared to normal tissue. However, a specific ligand for LGR5 has not yet been determined, and the function is still not clear. An LGR5-specific monoclonal antibody (mAb) is needed as a tool for detection and analysis of LGR5 biological function and cancer therapy. To date, no mAb against LGR5 that retains high affinity and specificity has been reported. Here, we report successful establishment and characterization of a mAb (KM4056) that specifically recognizes the extracellular N-terminal domain of human LGR5, but not LGR4 or LGR6. This mAb has potent complement-dependent cytotoxicity (CDC) activity in vitro and shows strong anti-tumor activity in vivo against xenograft model by transplanting LGR5 expressing CHO transfectants into SCID mice. Thus, KM4056 can be a useful tool for detection of LGR5 positive cells and analysis of LGR5 biological function.

The Current Status and Prospects of Antibody Engineering for Therapeutic Use: Focus on Glycoengineering Technology

Monoclonal antibodies have demonstrated enormous potential as new classes of drugs that confer great benefits to patients, and more than 40 therapeutic antibodies have already been approved for clinical use. In particular, the past 5 years might be recognized as the period guiding the new era for engineered antibodies, with the successful approval of numerous antibody-drug conjugates, bispecific antibodies, and glyco-engineered antibodies for clinical applications. In this review, we summarize the development of antibody engineering technologies that are proving their concepts in the clinic, mainly focusing on the latest trends in defucosylated antibody technologies.

Engineered anti‐CD20 antibodies with enhanced complement‐activating capacity mediate potent anti‐lymphoma activity

One of the major issues in current antibody therapy is insufficient efficacy. Various biological factors relating to the host’s immune system or tumor cells have been suggested to reduce the efficacy of anti‐CD20 therapy in B‐cell malignancies. In this study, we characterized the in vitro anti‐lymphoma activity of anti‐CD20 antibodies having a novel engineered heavy chain with enhanced complement‐dependent cytotoxicity (CDC). Anti‐CD20 antibodies having a variant heavy constant region of mixed IgG1/IgG3 isotype, which have previously been found to enhance CDC, were investigated for their in vitro CDC against lymphoma cells and whole blood B‐cell depletion activity. Use of the variant constant region greatly increased the CDC of an anti‐CD20 antibody having variable regions identical to those of rituximab to the level shown by an IgG1 antibody of ofatumumab. Although the whole blood assay showed different cytotoxicity patterns among individual blood donors, the CDC‐enhancing variant of rituximab showed higher activity than the parent IgG1 and consistently showed maximized activity when further combined with antibody‐dependent cellular cytotoxicity (ADCC)‐enhancing modification by fucose removal from Fc‐linked oligosaccharides. In addition, the rituximab variant showed potent CDC against transfectant cells with lower CD20 expression and chronic lymphocytic leukemia–derived cell lines with higher complement regulatory proteins. These findings suggest that CDC enhancement, both alone and in combination with ADCC enhancement, increases the anti‐lymphoma activity of anti‐CD20 antibodies irrespective of individual differences in effector functions, and renders current anti‐CD20 therapy capable of overcoming the potential resistance mechanisms. (Cancer Sci 2009; 100: 2411–2418)

One-step induction of neurons from mouse embryonic stem cells in serum-free media containing vitamin B12 and heparin.

We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B12 and heparin, but without any expensive cytokines. After 14 days in culture, β-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B12/heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B12/heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B12/heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinsons disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.

Nonfucosylated anti-CD20 antibody potentially induces apoptosis in lymphoma cells through enhanced interaction with FcγRIIIb on neutrophils

We demonstrate herein the augmentation of rituximab-mediated apoptosis in lymphoma cell lines by cross-linking with recombinant FcgammaRs, which is further enhanced by using a nonfucosylated variant of rituximab having strong FcgammaRIII-binding capacity. Furthermore, we show that neutrophils can serve as physiological cross-linkers that augment anti-CD20-mediated apoptosis, as evidenced by (i) the neutrophil-augmented apoptosis was more profound for the nonfucosylated variant of rituximab and (ii) the mechanism depended on FcgammaRIIIb but not on FcgammaRIIa. Taken together, we suggest a potential anti-tumour mechanism of nonfucosylated anti-CD20 antibody by which antibody molecules are cross-linked through enhanced interaction with FcgammaRIIIb in neutrophils.

Allergic diseases: From bench to clinic - Contribution of the discovery of interleukin-5

T helper 2 cells produce a number of cytokines including inteleukin (IL)-5, IL-4 and IL-13. Group 2 innate lymphoid cells (ILC2s) also produce IL-5 under sterile conditions. IL-5 is interdigitating homodimeric glycoprotein and a member of the four α helical bundle motifs conserved among hematopoietic cytokines. IL-5 exerts its effects on target cells via IL-5 receptor (IL-5R), composed of an IL-5R α and βc subunit. The membrane proximal proline-rich motif of the cytoplasmic domain of both IL-5R α and βc subunits is essential for IL-5 signal transduction. Although IL-5 was initially identified by its ability to support the growth and terminal differentiation of mouse B cells into antibody-secreting cells, recombinant IL-5 exerts pleiotropic activities on various target cells. For example, IL-5 is now recognized as the major maturation and differentiation factor for eosinophils in mice and humans. Overexpression of IL-5 in mouse significantly increases eosinophil numbers and antibody levels in vivo, while mice lacking a functional gene for IL-5 or IL-5R display developmental and functional impairments in B cell and eosinophil lineages. In mice, the role of the IL-5/IL-5R system in the production and secretion of Immunoglobulin (Ig) M and IgA in mucosal tissues has been reported. Although eosinophils protect against invading pathogens including virus, bacteria and helminthes, they are also involved in the pathogenesis of various diseases, such as food allergy, asthma, and inflammatory bowel diseases. The recent expansion in our understanding in the context of IL-5 and IL-5-producing ILC2s in eosinophil activation and the pathogenesis of eosinophil-dependent inflammatory diseases has led to advances in therapeutic options. A new therapy currently under invetigarion in clinical trials uses humanized monoclonal antibodies against IL-5 or the IL-5R. In this review, we summarize our current understanding of the functions of IL-5 and its receptor, the innate regulation of IL-5-producing cells, and therapeutic potential of anti-IL-5 and anti-eosinophil (IL-5R) antibodies.