Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Abstract

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.

CRISPR/Cas9-Based Genome Editing and Its Application in Aspergillus Species

“…For instance, adalimumab, a human anti-TNFα antibody, was produced by fusing it with AmyB, a α-amylase. Then, CRISPR/Cas9-based genome editing was used to delete the Aooch1 encoding a key enzyme of hyper-mannosylation process, to assess the recombinant antibody’s capacity to bind to FcγRIIIa [ 90 ]. This genome editing system was used to investigate the functional characterization of glycerol dehydrogenase, revealing that AoGld3, a glycerol dehydrogenase, is involved in the production of the secondary metabolite kojic acid by influencing the expression of kojA (an enzyme gene) and kojR (a transcription factor gene) involved in the kojic acid biosynthesis [ 91 ].…”

Advances in Genetic Engineering Technology and Its Application in the Industrial Fungus Aspergillus oryzae

“…This study showed that the repeatable marker-free genome editing approach can effectively delete or integrate multiple genes in the industrial A. oryzae strain ( Katayama et al, 2019 ). In the production experiment of human antibody (anti-TNFα antibody adalimumab) by A. oryzae , the CRISPR/Cas9 technique was used for the deletion of the Aooch1 gene, which encodes a key enzyme for the hypermannosylation process, to detect the binding ability of the recombinant antibody with FcγRIIIa ( Huynh et al, 2020 ). In addition, the CRISPR/Cas9 system was also used to delete AoGld3 , which is one of 45 glycerol dehydrogenase genes in A. oryzae , to investigate its effect on the production of the secondary metabolite kojic acid.…”

The ancient koji mold (Aspergillus oryzae) as a modern biotechnological tool

“…Adalimumab has been used in the treatment of some chronic inflammatory diseases such as rheumatoid arthritis. Production of adalimumab was successfully conducted in the culture supernatant of A. oryzae transformants with comparable affinities and bioactivities to its commercial counterpart (Huynh et al 2020).…”