Efficient Production of Antibody Fragments by the Filamentous Fungus Trichoderma reesei

Abstract

We have engineered the filamentous fungus Trichoderma reesei to assemble and secrete immunologically authentic engineered Fab antibody fragments into the culture medium. A major improvement in yield was achieved by fusing the heavy Fd chain to the T. reesei cellulase, CBHI. The yields of secreted, immunologically active Fab and CBHI-Fab fusion were 1 mg/l and 150 mg/l, respectively. The Fab fragment can be released from the fusion protein CBHI-Fab by an extracellular T. reesei protease. There was no detectable difference in affinity for the antigen between the engineered Fab and the idiotypic antibody.

Characterization of Humanized Antibodies Secreted byAspergillus niger

“…This is the system that we adopted in this work. An Fab fragment of an antibody was previously produced in T. reesei (19), and higher titers (up to 150 mg/liter) of the secreted fragment were obtained when the heavy chain was produced as a fusion with the native cellobiohydrolase I protein. However, in that study, the light chain was not produced as a fusion.…”

“…This approach has been used by Frenken et al (7) for the production of an ScFv fragment in A. niger. Work with T. reesei has demonstrated the production and assembly of an Fab fragment; in this work, the heavy chain (Fd) was expressed as a fusion with native secreted cellobiohydrolase I, while the light chain was not expressed as a fusion protein (19). Titers of up to 150 mg of secreted Fab/liter were observed.…”

Regulation of secreted protein production by filamentous fungi: recent developments and perspectives

“…The cbhl locus of T. reesei appears to be one of high transcriptional activity and has been used successfully to target heterologous gene expression (Harkki et al, 1991;Nyyssonen et al, 1993). The high levels of glucoamylase produced by A .…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…108 Indeed recently, it has been shown that the high heterogeneity observed between transformed clones can also be observed between genetically identical cells in the same been produced at high titer in the range of 1 g/l for IgG and Fab. 124,125 No difference was observed in antigen binding affinity, pharmacokinetic or ADCC. However immunoglobulins were produced as a mixture of glycosylated and aglycosylated ADCC activity of anti-CD20 recombinant antibody.…”