[Show abstract][Hide abstract] ABSTRACT: 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 (FcγR) 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 FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-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 FcγRIIIa 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 FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.
Journal of Virology 03/2012; 86(11):6189-96. DOI:10.1128/JVI.00491-12 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the 2000s, therapeutic antibodies have been shown to improve overall survival as well as time to disease progression in a variety of human malignancies such as breast, colon, and hematological cancers. In 1980s, the administration of nonhuman therapeutic antibodies, such as mouse monoclonal immunoglobulin, caused a severe host immune response, referred to as the human antimouse antibody (HAMA) response, which resulted in the prompt elimination of theadministered therapeutics as a foreign substance from human patients before a sufficient therapeutic effect was achieved. Recent progress in antibody engineering technology has overcome this major problem of immunogenicity. Mouse/human chimeric and complementarity-determining region (CDR)-grafted antibodies have been generated as humanized antibodies, and fully humanized antibodies have also been generated from transgenic mice capable of producing human antibodies, as well as from phage libraries of human immunoglobulin; antibodies produced in both manners are of sufficiently low immunogenicity to be applied clinically.
[Show abstract][Hide abstract] ABSTRACT: Human leukocyte receptor IIIa (Fc gamma RIIIa) 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 Fc gamma RIIIa binding, allowing the antibodies to enhance dramatically the antibody effector functions of ADCC. In this study, the contribution of Fc gamma RIIIa oligosaccharides to the strength of the Fc gamma RIIIa/antibody complex was analyzed using a serial set of soluble human recombinant Fc gamma RIIIa lacking the oligosaccharides. A nonfucosylated antibody IgG1 appeared to have a significantly higher affinity to the wild-type Fc gamma RIIIa 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 Fc gamma RIIIa glycosylation was removed. Our gain-of-function analysis in the Fc gamma RIIIa 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 Fc gamma RIIIa modified by N-162 alone showed a significantly higher binding affinity to nonfucosylated IgG1 than did the wild-type Fc gamma RIIIa. Attachment of the other four oligosaccharides, especially the Fc gamma RIIIa oligosaccharide at Asn-45 (N-45), hindered the high binding affinity of Fc gamma RIIIa to nonfucosylated IgG1. Our data clearly demonstrated that N-45 is an inhibitory element for the high Fc gamma RIIIa binding affinity mediated by N-162 to nonfucosylated antibodies. This information can be exploited for the structural-based functional study of Fc gamma RIIIa.
[Show abstract][Hide abstract] ABSTRACT: 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((R))) and anti-Her2/neu IgG1 trastuzumab (Herceptin((R))). ADCC is triggered upon the binding of lymphocyte receptors (FcgammaRs) 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 alpha-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.
[Show abstract][Hide abstract] ABSTRACT: Currently, removal of core fucose from the Fc oligosaccharides of therapeutic antibodies is widely recognized as being of great importance for the effector function of antibody-dependent cellular cytotoxicity, and alpha-1,6-fucosyltransferase (FUT8) knockout cells have been generated as an ideal host cell line for manufacturing such therapeutics. Here, we attempted to identify genes other than FUT8 that could be targeted for the manufacture of non-fucosylated therapeutics. Loss-of-function analyses using siRNAs against three key genes involved in oligosaccharide fucosylation in Chinese hamster ovary (CHO) cells revealed that there was a positive correlation between the Fc oligosaccharide fucosylation and the mRNA expression through the origin in the cases of both GDP-fucose 4,6-dehydratase (GMD) and FUT8, but not for the GDP-fucose transporter, suggesting that there is no functional redundancy in GMD and FUT8. GMD knockout CHO/DG44 cells were successfully established, and were confirmed to be devoid of intracellular GDP-fucose and to produce completely non-fucosylated antibodies. GMD knockout cells recovered their fucosylation capability through the salvage pathway upon addition of l-fucose into the culture medium, and exhibited equable morphology, growth kinetics and recombinant protein productivity, demonstrating that loss of oligosaccharide fucosylation has no impact on these cellular phenotypes. Our results demonstrate that GMD knockout is a new strategy applicable to the manufacture of non-fucosylated therapeutic antibodies, and completely O-fucose-negative therapeutics as well.
Journal of Biotechnology 07/2007; 130(3):300-10. DOI:10.1016/j.jbiotec.2007.04.025 · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.
Biotechnology and Bioengineering 07/2006; 94(4):680-8. DOI:10.1002/bit.20880 · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To generate industrially applicable new host cell lines for antibody production with optimizing antibody-dependent cellular cytotoxicity (ADCC) we focused on the most important carbohydrate structure "fucose residues attached to the innermost GlcNAc residue of N-linked oligosaccharides via �. -1,6 linkage" (Shields, 2002; Shinkawa, 2003), and succeeded in disrupting both FUT8 (�. -1,6-fucosyltransferase gene) alleles in Chinese hamster ovary (CHO) cell line by sequential homologous recombination. FUT8-/- cell lines have morphology and growth kinetics similar to those of the parent. Antibodies produced by the engineered CHO cell lines strongly bound to human Fc�� receptor IIIa (Fc�� RIIIa) and showed approximately two orders of magnitude higher ADCC than anti- CD20 antibodies (Rituxan TM ) produced by parental cell lines without changing antigen-binding and complement-dependent cytotoxicity (CDC). Moreover, the engineered cell line remains stable, producing completely- defucosylated antibody with fixed quality and efficacy even in serum-free fed-batch culture. Thus, our approaches provide a new strategy for controlling the glycosylation profile of therapeutic recombinant proteins and could be a considerable advantage for the manufacture of glycoprotein therapeutics, especially antibodies.
[Show abstract][Hide abstract] ABSTRACT: We explored the possibility of converting established antibody-producing cells to cells producing high antibody-dependent cellular cytotoxicity (ADCC) antibodies. The conversion was made by constitutive expression of small interfering RNA (siRNA) against alpha1,6 fucosyltransferase (FUT8). We found two effective siRNAs, which reduce FUT8 mRNA expression to 20% when introduced into Chinese hamster ovary (CHO)/DG44 cells. Selection for Lens culinaris agglutinin (LCA)-resistant clones after introduction of the FUT8 siRNA expression plasmids yields clones producing highly defucosylated (approximately 60%) antibody with over 100-fold higher ADCC compared to antibody produced by the parental cells (approximately 10% defucosylated). Moreover, the selected clones remain stable, producing defucosylated antibody even in serum-free fed-batch culture. Our results demonstrate that constitutive FUT8 siRNA expression can control the oligosaccharide structure of recombinant antibody produced by CHO cells to yield antibodies with dramatically enhanced ADCC.
Biotechnology and Bioengineering 12/2004; 88(7):901-8. DOI:10.1002/bit.20326 · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.