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 ␣1,6-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. Antibody-dependent cellular cytotoxicity (ADCC),1 a lytic attack on antibody-targeted cells, is triggered upon binding of lymphocyte receptors (Fc␥Rs) to the constant region (Fc) of the antibodies. ADCC is considered to be a major function of some of the therapeutic antibodies, although antibodies have multiple therapeutic functions (e.g. antigen binding, induction of apoptosis, and complement-dependent cellular cytotoxicity) (1, 2).One IgG molecule contains two N-linked oligosaccharide sites in its Fc region (3). The general structure of N-linked oligosaccharide on IgG is complex-type, characterized by a mannosyl-chitobiose core (Man3GlcNAc2-Asn) with or without bisecting GlcNAc/L-fucose (Fuc) and other chain variants including the presence or absence of Gal and sialic acid. In addition, oligosaccharides may contain zero (G0), one (G1), or two (G2) Gal.Recent studies have shown that engineering the oligosaccharides of IgGs may yield optimized ADCC. ADCC requires the presence of oligosaccharides covalently attached at the conserved Asn 297 in the Fc region and is sensitive to change in the oligosaccharide structure. In the oligosaccharide, sialic acid of IgG has no effect on ADCC (4). The relationship between the Gal residue and ADCC is controversial. Boyd et al. (4) have shown that obvious change was not found in ADCC after removal of ...

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.

Human leukocyte receptor IIIa (FcγRIIIa) plays an important role in mediating therapeutic antibodies’ antibody-dependent cellular cytotoxicity (ADCC), which is closely related to the clinical efficacy of anticancer processes in humans in vivo. The removal of the core fucose from oligosaccharides attached to the Fc region of antibodies improves FcγRIIIa binding, allowing the antibodies to enhance dramatically the antibody effector functions of ADCC. In this study, the contribution of FcγRIIIa oligosaccharides to the strength of the FcγRIIIa/antibody complex was analyzed using a serial set of soluble human recombinant FcγRIIIa lacking the oligosaccharides. A nonfucosylated antibody IgG1 appeared to have a significantly higher affinity to the wild-type FcγRIIIa fully glycosylated at its five N-linked oligosaccharide sites than did the fucosylated IgG1, and this increased binding was almost abolished once all of the FcγRIIIa glycosylation was removed. Our gain-of-function analysis in the FcγRIIIa oligosaccharide at Asn-162 (N-162) confirmed that N-162 is the element required for the high binding affinity to nonfucosylated antibodies, as previously revealed by loss-of-function analyses. Interestingly, beyond our expectation, the FcγRIIIa modified by N-162 alone showed a significantly higher binding affinity to nonfucosylated IgG1 than did the wild-type FcγRIIIa. Attachment of the other four oligosaccharides, especially the FcγRIIIa oligosaccharide at Asn-45 (N-45), hindered the high binding affinity of FcγRIIIa to nonfucosylated IgG1. Our data clearly demonstrated that N-45 is an inhibitory element for the high FcγRIIIa binding affinity mediated by N-162 to nonfucosylated antibodies. This information can be exploited for the structural-based functional study of FcγRIIIa.

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (Fc␥R) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of Fc␥R binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to Fc␥R-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque Fc␥RIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that Fc␥R-mediated activities distinct from Fc␥RIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.

Leinamycin (1) was isolated from the culture broth of a Streptomyces sp. in 1989, 1-3 and its structure was elucidated by spectroscopic analysis, 1 X-ray crystallography, 4 and chemical synthesis. 5 This antibiotic contains an unusual 1,3-dioxo-1,2dithiolane moiety, which is connected to the 18-membered lactam through a spiro linkage, and appeared to be a new class of natural product. 1 exhibited significant antitumor activity in some murine tumor models. 2 We previously reported that 1 induces single-strand scission of DNA in the presence of thiol cofactors in vitro. We report here the detailed chemistry of thiol-activation and DNA-cleavage induced by 1.Addition of 1.5 equiv of 2-mercaptoethanol (2-ME) to a solution of 1 in MeOH/10 mM phosphate buffer (pH 7) (1/9, v/v) resulted in a rapid conversion to a major product with several minor products. Other thiols including ethanethiol, dithiothreitol, cysteine, and glutathione afforded approximately equal amounts of 2. We isolated 2 from the reaction mixture with reverse-phase HPLC (70%, yield), but the full characterization of 2 failed due to its instability in DMSO. Treatment of 2 with K 2 CO 3 and iodomethane afforded a stable methyl ester 2a (68%, yield). Characterization of 2a by 1D and 2D-NMR experiments established an unexpected structure, in which the 1,3-dioxo-1,2-dithiolane moiety and the 6,7-olefin were missing and a new 3,7-sulfide linkage and 6-hydroxyl group were observed. Treatment of 1 with 2-ME in a MeOH-rich solvent, MeOH/0.5 M phosphate buffer (pH 7) (99/1, v/v), afforded the methanol adduct 2b 6 as the main product.After reaction of 1 with calf thymus DNA in the presence of 2-ME (drug/DNAbp/2ME ) 1/20/1.5), the DNA was purified by ethanol precipitation. The DNA showed a UV spectrum characteristic of the complex with a chromophore. Although it was stable at 4°C, gradual release of the chromophore from the DNA was observed at 37°C. The rate and efficiency of the release increased with further an increase in the temperature. On a preparative scale, we isolated the released chromophore 3 from the leinamycin-treated calf thymus DNA (75% yield from 1). In the 13 C NMR spectrum of 3 all of the resonances were comparable to those of 2, and five additional resonances were found, suggesting the addition of a purine residue to 2. In the 1 H-NMR spectrum, all of the resonances were also comparable to those of 2. One additional nonexchangeable resonance at 7.77 ppm (1H) was found. The only nucleobase that gives one nonexchangeable resonance is guanine. Observation of the NOE between the guanine H-8 and the 6-CH 3 is consistent with alkylation of the C-6 carbon by the N-7 of guanine. These spectroscopic data revealed that 3 is a leinamycin-N7 guanine adduct. This was supported by the molecular formula that was established as the sodium salt C 27 H 31 O 7 N 7 S 2 Na: HRFABMS (M + Na) + m/z 652.1600, calcd 652.1624. 1 did not react with guanosine nucleotide monomer or single stranded DNA, suggesting that the alkylation of DNA could be attributable to the unique...