The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

Pereira, Natasha Ann; Chan, Kah Fai; Lin, Po-Chuan; Song, Zhiwei

doi:10.1080/19420862.2018.1466767

Cited by 267 publications

(229 citation statements)

References 148 publications

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“…However, core-fucosylation is likely most known for its capacity to adjust the humoral immune response by altering antibody-dependent cellular cytotoxicity (ADCC). Thus, different strategies for reducing core-fucosylation in therapeutic monoclonal antibodies have been developed to enhance ADCC 8 . Notably, FUT8 is upregulated in numerous types of cancer, suggesting that blocking its activity could be a promising strategy for improving antitumor immune responses 9 .…”

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confidence: 99%

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

et al. 2020

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Core-fucosylation is an essential biological modification by which a fucose is transferred from GDP-β-L-fucose to the innermost N-acetylglucosamine residue of N-linked glycans. A single human enzyme α1,6-fucosyltransferase (FUT8) is the only enzyme responsible for this modification via the addition of an α-1,6-linked fucose to N-glycans. To date, the details of substrate recognition and catalysis by FUT8 remain unknown. Here, we report the crystal structure of FUT8 complexed with GDP and a biantennary complex N-glycan (G0), which provides insight into both substrate recognition and catalysis. FUT8 follows an S N 2 mechanism and deploys a series of loops and an α-helix which all contribute in forming the binding site. An exosite, formed by one of these loops and an SH3 domain, is responsible for the recognition of branched sugars, making contacts specifically to the α1,3 arm GlcNAc, a feature required for catalysis. This information serves as a framework for inhibitor design, and helps to assess its potential as a therapeutic target.

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confidence: 99%

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

et al. 2020

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“…Another important Fc-mediated immune effector function that plays the most important role in depleting tumor cells is called antibody-dependent cellular cytotoxicity (ADCC). It has been proved previously that the absence of core fucose on Fc N-glycan structures can lead to enhanced ADCC activity (23, 24). It has also been confirmed that apart from afucosylation, galactosylation levels could also influence ADCC activity; however, the role of afucosylation is more prominent.…”

Section: Resultsmentioning

confidence: 99%

Implementation of Design of Experiments (DOE) for Optimization of Feeding Strategy and Glyco-Engineering of Trastuzumab Biosimilar

Mahboudi¹,

Samavat²,

Afrah³

et al. 2019

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2Fed-batch cell culture is the most commonly used process for antibody production in 3 biopharmaceutical industries. Basal media, feed, feeding strategy and glycan structures are always 4 among the most important concerns during process development and optimization. In this study, 5 first, a traditional screening study was performed to identify the top media/feed combinations by 6 evaluating the cell culture performance including cell growth and protein titre. Optimization of the 7 process was also performed using response surface methodology in order to find the most optimum 8 feeding strategy and glucose set point regarding final titre of the recombinant monoclonal antibody 9 being produced in Chinese hamster ovary cell line. The focus of this study is not only on titre, but 10 also on product quality and comparability especially protein glycosylation. The prediction model 11 of product titre as a function of feeding percentage and glucose set point was successfully applied 12 for the second set of experiments that was performed for glycan improvement. Statistical design 13 of experiments was applied to determine the most important factors and their effects on 14 galactosylated and afucosylated glycans. Uridine, manganese, galactose and fucosyltransferase 15 inhibitor were chosen to evaluate if their presence can affect glycans and to obtain their best 16 combination for fed-batch culture supplementation. We determined that 2.5 % daily feeding 17 combined with maintaining the glucose set point on 2.5±0.2 g/L could achieve final titre of 2.5± 18 0.1 g/L. Galactosylation of antibody was increased about 25% using MnCl 2 and galactose while 19 afucosylation was increased about 8% in presence of fucosyltransferase inhibitor. Galactose and 20 Mn 2+ led to a shift from G0F to G1F and presence of Fucosyltransferase inhibitor caused to an 21 increase in G0 compared to its absence. These results demonstrated that supplementation of culture 22 with all these components can provide exact control of antibody galactosylation and fucosylation 23 with minimal impact on culture characteristics and product quality attributes. Subsequently, 24 validation experiments were also carried out in 5L STR bioreactors which showed that similar 25 results could be achieved in bioreactors compared to shake flasks regarding both titre and quality. 26 Keywords: Trastuzumab biosimilar, feeding strategy optimization, design of 27 experiments (DOE), galactosylation, afucosylation 3 28 Abbreviations 29 mAb: Monoclonal antibody; DOE: design of experiment; RSM: response surface 30 methodology; CDC: complement dependent cytotoxicity; ADCC: antibody dependent cell 31 cytotoxicity; CQA: critical quality attributes; Fc: fragment crystallizable; FTI: fucosyl transferase 32 inhibitor; CHO: Chinese hamster ovary; HER2; human epidermal growth factor receptor 2; CCD: 33 central composite design; ANOVA: analysis of variance; HPLC: high pressure liquid 34 chromatography; STR : stirred tank reactor; DO: Dissolved oxygen; VCD: viable cell density; 35 VCC: viabl...

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“…The mutation abrogating the N-glycosylation at Asn in the Fc region aborts the interaction with FcγRIIIa [50]. The fucosylated antibody was reported to reduce the binding a nity to FcγRIIIa [51]. In Humira ® , the fucosylated N-glycan is contained with the ratio of 10 % [52], and thus relatively low a nity to FcγRIIIa was detected when high concentrations of Humira ® (15 µg/ml) and TNFα (40 ng/ml) were used (Fig.…”

Section: Discussionmentioning

confidence: 97%

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Huynh

Morita

Sakamoto

et al. 2020

Preprint

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Background: Monoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNFα antibody adalimumab, one of the world’s best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa.Results: Adalimumab was expressed in A. oryzae by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions.Conclusion: These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient’s welfare.

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The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

Cited by 267 publications

References 148 publications

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

Implementation of Design of Experiments (DOE) for Optimization of Feeding Strategy and Glyco-Engineering of Trastuzumab Biosimilar

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

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