Secretion of functional antibody and Fab fragment from yeast cells.

Horwitz, Arnold H.; Chang, Chia-Ching; Better, Marc; Hellström, Karl Erik; Robinson, RR

doi:10.1073/pnas.85.22.8678

Cited by 154 publications

(58 citation statements)

References 27 publications

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“…Although there were previous reports of secretion of fulllength antibody by yeast cells (11,32), the titers attained were much lower than those that we report here. We observed higher titers of secreted trastuzumab (up to 0.9 g/liter) than of Hu1D10 (0.2 g/liter).…”

Section: Discussioncontrasting

confidence: 54%

“…Wood et al (32) showed that glycosylated and nonglycosylated forms of heavy chain were produced in yeast cells. Similarly, Horwitz et al (11) showed that heavy chain ␥ produced in yeast cells appeared as a doublet on reducing SDS-PAGE, although the glycosylation states of the two heavy-chain forms were not determined. We were able to produce an aglycosylated mutant form of trastuzumab by changing the Asn residue at position 297 to Gln, but the titers of this form of the secreted antibody were reduced.…”

Section: Discussionmentioning

confidence: 99%

“…Secretion of ScFv fragments into the culture supernatant of Pichia pastoris at high titers, e.g., 250 mg/liter and 1.2 g/liter, has been reported by Eldin et al (6) and by Freyre et al (8), respectively, and up to 40 mg of secreted Fab/liter has been achieved with this yeast (16,25). In comparison to antibody fragments, there are very few reports of the production of full-length antibodies in microbial systems and, when reported, the titers have been very low (11,32). However, Simmons et al (23) recently reported the periplasmic accumulation of assembled, full-length immunoglobulin G1 (IgG1) in E. coli at titers of up to 150 mg/liter.…”

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confidence: 99%

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Characterization of Humanized Antibodies Secreted byAspergillus niger

Ward¹,

Lin²,

Victoria³

et al. 2004

Appl Environ Microbiol

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Two different humanized immunoglobulin G1( ) antibodies and an Fab fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex 6 GlcNAc 2 to Hex 15 GlcNAc 2 . An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.

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Section: Discussioncontrasting

confidence: 54%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of Humanized Antibodies Secreted byAspergillus niger

Ward¹,

Lin²,

Victoria³

et al. 2004

Appl Environ Microbiol

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“…Some reports have addressed the production of antibody by yeast, although in those studies, productivity was either low or not mentioned (15,20,25,30,44). For the production of antibodies in yeast, it will be necessary to increase productivity.…”

Section: Discussionmentioning

confidence: 99%

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

Kuroda

Kobayashi

Kitagawa

et al. 2008

Appl Environ Microbiol

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When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.Antibodies for pharmaceutical applications have recently received a great deal of attention due to their specific antigenbinding activities, antibody-dependent cellular cytotoxicity, and other useful characteristics. Indeed, sales of antibodies for pharmaceuticals have grown rapidly over the past decade and are expected to exceed 30 billion U.S. dollars by 2010 (1). A large volume of antibodies is required for pharmaceuticals, as antibodies are primarily marketed for chronic conditions. Antibodies possess relatively low potency, which results in the need for accumulating doses over time (10). Although therapeutic antibodies have great potential value, they have also been viewed with some skepticism. Antibodies are expensive to produce, in part because they are commonly manufactured by using batch/fed-batch cultures of mammalian cells. The increasing demand to reduce the cost of antibody production has promoted research into the development of an antibody expression system that is more practical than expression with mammalian cells.Development of transgenic plants and animals as alternative hosts is therefore a promising field of study. Monoclonal antibodies have, thus far, been successfully produced from a number of sources, including plants, the milk of transgenic goats, the eggs of transgenic chickens, etc. (4, 12, 32). Moreover, antibodies derived from certain newly developed transgenic systems share physical characteristics that are similar to those of antibodies from mammalian cells such as Chinese hamster ovary (CHO) cells while exhibiting higher antibody-dependent cellular cytotoxicity activity due to the absence of fucose residues in N-linked sugar chains (6, 45). These alternative transgenic expression systems could reduce the cost of large-scale antibody production. However, the prolonged construction of transgenics remains a major disadvantage in terms of market demands.The production of antibodies and antibody fragments has been studied by using various microorganism expression systems, including Escherichia coli, fungus, and yeast (7,13,37,41). Since microorg...

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“…While an efficient ADCC activity was observed, recombinant IgG1 failed to induce CDC. 113 A 180-fold improvement in secretion was obtained by using a mutant of α mating factor 1 leader peptide and by co-expressing protein disulfide isomerase (PDI). 114 Evans et al recently reported the production at high yield (40-90 mg/l) of scFv fused to human serum albumin.…”

mentioning

confidence: 99%