Expression of an Anti-CD3 Single-Chain Immunotoxin with a Truncated Diphtheria Toxin in a Mutant CHO Cell Line

Liu, Yuan Yi; Gordienko, Irina; Mathias, Askale; Ma, Shenglin; Thompson, Jeffrey P.; Woo, Jeong Soo; Neville, David M.

doi:10.1006/prep.2000.1255

Cited by 34 publications

(35 citation statements)

References 36 publications

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“…Based on our understanding of the processing and secretion of ␣-factor in S. cerevisiae (37), we expect that after entering the endoplasmic reticulum (ER), the preproimmunotoxin undergoes initial processing to remove the presequence, followed by folding and N-linked glycosylation at the three potential sites in the prosequence region. Potential glycosylation sites of the immunotoxin have been removed (22). The proimmunotoxin could then be transported from the ER to the Golgi complex, where it might undergo further modification of oligosaccharide chains before it is finally processed to yield the immunotoxin by proteolytic cleavage at the Kex2 site introduced between the prosequence and the immunotoxin.…”

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confidence: 99%

Overexpression of an Anti-CD3 Immunotoxin Increases Expression and Secretion of Molecular Chaperone BiP/Kar2p byPichia pastoris

Liu

Woo

Neville

2005

Appl Environ Microbiol

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We previously reported that the secretory capacity of Pichia pastoris is limited with respect to the secretion of a 96.5-kDa bivalent anti-CD3 immunotoxin; double-copy expression generated more translation products than single-copy expression but did not increase the secretion of the immunotoxin. In Saccharomyces cerevisiae heterologous protein secretion has been reported to increase the expression of molecular chaperones, most prominently BiP/Kar2p. We therefore investigated the relationships between immunotoxin secretion and Kar2p expression in P. pastoris. We found that expression of the immunotoxin in P. pastoris increased the expression of Kar2p to levels that surpassed the retrieval capacity of the cell, leading to secretion of Kar2p into the medium. The level of Kar2p secretion was correlated with the copy number of the immunotoxin gene. Intracellular Kar2p was found to bind exclusively to the unprocessed immunotoxin containing the prosequence of ␣-factor in the endoplasmic reticulum. These results show that Kar2p is intimately involved in immunotoxin secretion in P. pastoris. The limited capacity of P. pastoris to retain a sufficiently high level of intracellular Kar2p may be a factor restricting the production of the immunotoxin.The yeast Pichia pastoris has been developed into a highly successful system for the production of recombinant proteins having different origins (5). However, the efficiency of heterologous secretion can vary widely among the expressed proteins. Some eukaryotic proteins, such as human serum albumin and insulin, can be secreted at very high levels (grams per liter), whereas the secretion levels of many other proteins are significantly lower or even undetectable.We have expressed a bivalent anti-CD3 immunotoxin in P. pastoris with a moderate secretion level (about 5 mg/liter in shake flask cultures and 37 mg/liter in fermentation cultures) (46,47). Attempts to increase the production of this protein by using protease inhibitors and protease-deficient strains and by increasing the copy number of the gene were not successful (21). Interestingly, strains with two copies of the immunotoxin gene generated more translation product, but there was not an increase in the secretion of the intact protein, indicating that the expressing host has a limited capacity to fold and/or secret the immunotoxin. Structurally, this multidomain immunotoxin has several interesting features (Fig. 1). The N terminus consists of the diphtheria toxin (DT) catalytic domain (DT A), followed by the toxin translocation domain ending at DT residue 390, followed by two tandem scFv domains separated by a (G 4 S)3 linker (40). The junction between the catalytic and translocation domains contains an accessible Kex2 cleavage site spanned by a disulfide loop. To facilitate secretion, the toxin gene is preceded by the Saccharomyces cerevisiae ␣-mating factor preprosequence. Based on our understanding of the processing and secretion of ␣-factor in S. cerevisiae (37), we expect that after entering the endoplasmic reticulum...

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confidence: 99%

Overexpression of an Anti-CD3 Immunotoxin Increases Expression and Secretion of Molecular Chaperone BiP/Kar2p byPichia pastoris

Liu

Woo

Neville

2005

Appl Environ Microbiol

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“…Furthermore, the bivalent immunotoxin production in an EF-2 mutant CHO cell expression system was limited to 5 mg/liter and could not be increased by selection for multiple gene insertions (Neville, unpublished data). Due to this limitation, except for three reports from our laboratory (12,20,25), all recombinant immunotoxin production for therapeutic uses has been limited to Escherichia coli production, which has required denaturation and refolding from inclusion bodies (6). However, refolding of the multidomain structure of the bivalent immunotoxin from E. coli was inefficient, and complete bioactivity was not recovered (25).…”

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Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

Woo

Liu

Stavrou

et al. 2004

Appl Environ Microbiol

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The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G 4 S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut ؉ ) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G 4 S) was developed for treatment of T-cell leukemia and autoimmune diseases and tolerance induction for transplantation. This immunotoxin selectively kills human T cells and is the most efficacious of a variety of anti-T-cell immunotoxins that have been developed (20). The bivalent immunotoxin contains the first 390 amino acid residues of diphtheria toxin (DT) and two tandem sFv molecules that are responsible for binding the immunotoxin to the CD3ε␥ subunit of the T-cell receptor complex on human T cells. The first 390 amino acid residues of DT (DT390) contain the catalytic domain or A chain of DT that inhibits protein synthesis by ADP ribosylation of EF-2 and the translocation domain that translocates the catalytic domain to the cytosol by interactions with cytosolic Hsp90 and thioredoxin reductase (17).The inhibition of protein synthesis by the catalytic domain makes it difficult to produce the toxin-related proteins in most eukaryotic cells (e.g., wild-type CHO cells, insect cells, and yeasts). The use of toxin-resistant eukaryotic cells can overcome the immunotoxin toxicity. However, selection and characterization of toxin-resistant eukaryotic cells are tedious, labor-intensive, ...

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“…Correct post-translational modifications can support functionality of eukaryoticderived proteins. Although expression of immunotoxins has been theoretically limited because the products are toxic to the producing cells as soon as they reach the cytosol, some groups have reported the successful synthesis of immunotoxins in eukaryotic cells in vitro and in vivo (Chen et al 1997;Vallera et al 2000;Liu et al 2000;Jia et al 2008). These researchers found that the transfected eukaryotic cells retained their viability while continuously secreting immunotoxin proteins.…”

Section: Discussionmentioning

confidence: 98%

Human mesenchymal stem cells-like cells as cellular vehicles for delivery of immunotoxin in vitro

Sun

et al. 2008

Biotechnol Lett

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Human mesenchymal stem cells-like cells (hMSCs-like cells) were used as a tumor treatment platform for the systemic delivery of immunotoxin genes. VEGF165-PE38 recombinant immunotoxin served as the model system. hMSCs-like cells were isolated, expanded, and electroporated with the pIRES2-VEGF165PE38-EGFP plasmid. RT-PCR and ELISA were used to confirm the expression of VEGF165-PE38 in the transfected hMSCs-like cells. These cells released 1390 +/- 137 pg VEGF165-PE38/10(4)cells over 48 h into the culture medium and the supernatant was capable of selectively killing human umbilical vein endothelial cells (HUVECs) and increasing apoptosis in these cells. In contrast, RPMI8226 was not inhibited by identical supernatants. Thus, these results lay the foundation for further studies on the potential role of hMSCs-like cells as a targeted therapeutic delivery vehicle for immunotoxins.

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Expression of an Anti-CD3 Single-Chain Immunotoxin with a Truncated Diphtheria Toxin in a Mutant CHO Cell Line

Abstract: ADP-ribosylating immunotoxins are generally expressed in

Cited by 34 publications

References 36 publications

Overexpression of an Anti-CD3 Immunotoxin Increases Expression and Secretion of Molecular Chaperone BiP/Kar2p byPichia pastoris

Overexpression of an Anti-CD3 Immunotoxin Increases Expression and Secretion of Molecular Chaperone BiP/Kar2p byPichia pastoris

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

Human mesenchymal stem cells-like cells as cellular vehicles for delivery of immunotoxin in vitro

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