Masako Wakitani

Engineered antibodies of IgG1/IgG3 mixed isotype with enhanced cytotoxic activities.

Enhancement of multiple effector functions of an antibody may be a promising approach for antibody therapy. We have previously reported that fucose removal from Fc-linked oligosaccharides greatly enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies. Here, we report a unique approach to enhance complement-dependent cytotoxicity (CDC), another important effector function of antitumor antibodies, by using engineered constant region of human IgG1/IgG3 chimeric isotypes. We systematically shuffled constant domains of IgG1 and IgG3 to generate a comprehensive set of mixed chimeric isotypes of anti-CD20 antibodies. Among these, the variant 1133, consisting of the CH1 and the hinge each from IgG1 and the Fc from IgG3, was unexpectedly found to exhibit markedly enhanced CDC that exceeded wild-type levels. However, it lacked protein A-binding capacity, an important feature for the industrial production. To eliminate this deficiency, a portion in COOH-terminal CH3 domain of 1133 was substituted with IgG1, resulting in full recovery of protein A binding without compromising the enhanced CDC and ADCC activities. The CDC-enhancing effect using a chimeric isotype was also shown in CD52 antigen/antibody system. The ADCC activity of the variants was also maximized by the absence of fucose from its carbohydrate structure, a phenomenon that has previously been observed for wild-type antibodies. Enhanced cytotoxicity of a variant was confirmed in a cynomolgus monkey model. These findings suggest that the variant antibodies with IgG1/IgG3 chimeric constant regions and nonfucosylated oligosaccharides that possess dual-enhanced cytotoxic functions may be an improvement for the next generation of therapeutic antitumor antibodies.

Nonfucosylated Therapeutic IgG1 Antibody Can Evade the Inhibitory Effect of Serum Immunoglobulin G on Antibody-Dependent Cellular Cytotoxicity through its High Binding to FcγRIIIa

Purpose: Recent studies have revealed that fucosylated therapeutic IgG1s need high concentrations to compensate for FcγRIIIa-competitive inhibition of antibody-dependent cellular cytotoxicity (ADCC) by endogenous human plasma IgG. Here, we investigated whether ADCC of nonfucosylated therapeutic IgG1 is also influenced by plasma IgG in the same way as fucosylated IgG1s. Experimental Design:Ex vivo ADCC upon CD20+ human B cells was induced by incubation of human whole blood with nonfucosylated and/or fucosylated anti-CD20 IgG1s rituximab, and quantified by measuring the remaining CD19+ human B cells using flow cytometry. Results: Nonfucosylated anti-CD20 showed markedly higher (over 100-fold based on EC50) ex vivo B-cell depletion activity than its fucosylated counterpart in the presence of plasma IgG. The efficacy of fucosylated anti-CD20 was greatly diminished in plasma, resulting in the need for a high concentration (over 1.0 μg/mL) to achieve saturated efficacy. In contrast, nonfucosylated anti-CD20 reached saturated ADCC at lower concentrations (0.01-0.1 μg/mL) with much higher efficacy than fucosylated anti-CD20 in all nine donors through improved FcγRIIIa binding. Noteworthy, the high efficacy of nonfucosylated anti-CD20 was inhibited by addition of fucosylated anti-CD20. Thus, the efficacy of a 1:9 mixture (10 μg/mL) of nonfucosylated and fucosylated anti-CD20s was inferior to that of a 1,000-fold dilution (0.01 μg/mL) of nonfucosylated anti-CD20 alone. Conclusions: Our data showed that nonfucosylated IgG1, not including fucosylated counterparts, can evade the inhibitory effect of plasma IgG on ADCC through its high FcγRIIIa binding. Hence, nonfucosylated IgG1 exhibits strong therapeutic potential through dramatically enhanced ADCC at low doses in humans in vivo.

Double knockdown of α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC

BackgroundAntibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies.ResultsFirstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. α1,6-fucosyltransferase (FUT8), GDP-mannose 4,6-dehydratase (GMD), and GDP-fucose transporter (GFT), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of FUT8 and GMD siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with GFT siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of FUT8 and GMD, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture.ConclusionOur results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of FUT8 and GMD in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies.

Controlling Fucosylation Levels of Antibodies with Osmolality During Cell Culture in Several Host Cell Lines

Since a cost of therapeutic Monoclonal antibodies (MAbs) is much higher than other compounds, it is critical to produce high-efficacy MAbs efficiently. One method is to increase the effectiveness of a MAb, which in turn affects antibody-dependent cellular cytotoxicity (ADCC) is related defucosylation level (deFuc%) of MAbs. Since deFuc% of MAbs must be regulated for their quality control, it leads to careful consideration of the type of host cell employed. Thus, it is quite important to grasp the effects of culture conditions on the deFuc% in each cell line for the launched on the market and development, except for like a Chinese hamster ovary cells (CHOs) with α-1,6-fucosyltransferase gene knock out (PotelligentTM, BioWa, USA). For the MAbs produced in a rat myeloma cells (YB2/0), we found that osmolality of the culture medium is the major determinant of the deFuc%. In addition, deFuc% was not affected by the type of osmolytes (NaCl, KCl, fucose, fructose, and mannitol). We succeeded in controlling the deFuc% of MAbs arbitrarily 45–85% by maintaining medium osmolality during cultures (perfusion and fed-batch). We found the same correlation between the deFuc% and the culture osmolality in NS0 and SP2/0 cells as the in the YB2/0 cells.

Controlling Fucosylation Levels of Antibodies with Osmolality during Cell Culture

Because defucosylation levels (deFuc%) of monoclonal antibodies (MAbs) must be regulated for quality control, it is quite important to analyze the factors that affect deFuc%. For the MAbs produced in the rat hybridoma cell line YB2/0, we found that osmolality of the culture medium is the major determinant of deFuc%. deFuc% was linearly correlated with osmolality, with r2 being as high as 0.92. In addition, deFuc% was not affected by the type of compound used for controlling osmolality (NaCl, KCl, fucose, fructose, creatine and mannitol). We succeeded in controlling deFuc% of MAbs by maintaining medium osmolality constant during cultures (for both perfusion and fed-batch cultures).