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

<p>Abstract</p> <p>Background</p> <p>Antibody-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 <it>in vivo</it>. 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.</p> <p>Results</p> <p>Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. α1,6-fucosyltransferase (<it>FUT8</it>), GDP-mannose 4,6-dehydratase (<it>GMD</it>), and GDP-fucose transporter (<it>GFT</it>), 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 <it>FUT8 </it>and <it>GMD </it>siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with <it>GFT </it>siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of <it>FUT8 </it>and <it>GMD</it>, 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.</p> <p>Conclusion</p> <p>Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of <it>FUT8 </it>and <it>GMD </it>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.</p

Two mechanisms of the enhanced antibody-dependent cellular cytotoxicity (ADCC) efficacy of non-fucosylated therapeutic antibodies in human blood

<p>Abstract</p> <p>Background</p> <p>Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical mechanisms underlying the clinical efficacy of therapeutic antibodies, especially anticancer antibodies. Therapeutic antibodies fully lacking the core fucose of the Fc oligosaccharides have been found to exhibit much higher ADCC in humans than their fucosylated counterparts. However, data which show how fully non-fucosylated antibodies achieve such a high ADCC in human whole blood have not yet been disclosed. The precise mechanisms responsible for the high ADCC mediated by fully non-fucosylated therapeutic antibodies, even in the presence of human plasma, should be explained based on direct evidence of non-fucosylated antibody action in human blood.</p> <p>Methods</p> <p>Using a human <it>ex vivo </it>B-cell depletion assay with non-fucosylated and fucosylated anti-CD20 IgG1s rituximab, we monitored the binding of the therapeutic agents both to antigens on target cells (target side interaction) and to leukocyte receptors (FcγR) on effector cells (effector side interaction), comparing the intensities of ADCC in human blood.</p> <p>Results</p> <p>In the target side interaction, down-modulation of CD20 on B cells mediated by anti-CD20 was not observed. Simple competition for binding to the antigens on target B cells between fucosylated and non-fucosylated anti-CD20s was detected in human blood to cause inhibition of the enhanced ADCC of non-fucosylated anti-CD20 by fucosylated anti-CD20. In the effector side interaction, non-fucosylated anti-CD20 showed sufficiently high FcγRIIIa binding activity to overcome competition from plasma IgG for binding to FcγRIIIa on natural killer (NK) cells, whereas the binding of fucosylated anti-CD20 to FcγRIIIa was almost abolished in the presence of human plasma and failed to recruit NK cells effectively. The core fucosylation levels of individual serum IgG1 from healthy donors was found to be so slightly different that it did not affect the inhibitory effect on the ADCC of fucosylated anti-CD20.</p> <p>Conclusion</p> <p>Our results demonstrate that removal of fucosylated antibody ingredients from antibody therapeutics elicits high ADCC in human blood by two mechanisms: namely, by evading the inhibitory effects both of plasma IgG on FcγRIIIa binding (effector side interaction) and of fucosylated antibodies on antigen binding (target side interaction).</p

Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC-1

<p><b>Copyright information:</b></p><p>Taken from "Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC"</p><p>http://www.biomedcentral.com/1472-6750/7/84</p><p>BMC Biotechnology 2007;7():84-84.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2216013.</p><p></p> line 32-05-12 (filled squares) was cultured as a control. Viable cell density (A, solid lines), antibody concentration in the culture supernatant (A, dotted lines), and cell viability (B) were analyzed in the fed-batch culture. The oligosaccharide structures of the final products from 32-05-12 (C), FG1 (D), and FG16 (E) were analyzed using MALDI-TOF MS. The relative composition of each peak is shown as the relative amount to the total amount of oligosaccharide detected

Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC-0

<p><b>Copyright information:</b></p><p>Taken from "Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC"</p><p>http://www.biomedcentral.com/1472-6750/7/84</p><p>BMC Biotechnology 2007;7():84-84.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2216013.</p><p></p>n or hygromycin resistance gene and a short hairpin siRNA expression cassette controlled by the human tRNApromoter (B or C). The siRNA tandem expression plasmid consisted of a hygromycin resistance gene and two short hairpin siRNA expression cassettes targeting and (D). The transcribed shRNAs and tRNA-shRNA fusion product were processed into siRNAs by Dicer

Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC-2

<p><b>Copyright information:</b></p><p>Taken from "Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC"</p><p>http://www.biomedcentral.com/1472-6750/7/84</p><p>BMC Biotechnology 2007;7():84-84.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2216013.</p><p></p>A). The antigen-binding activity of the antibody was measured by ELISA (B). Antibody purified from the serum-free fed-batch cultures of cells transformed by the and tandem siRNA expression vector, FG1 (open circles), FG16 (open squares), and the parental cell line 32-05-12 (filled circles) are shown. Cytotoxicity (%) and absorbance are indicated as the mean values ± SD of triplicates

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