High Level Escherichia coli Expression and Production of a Bivalent Humanized Antibody Fragment

Carter, Paul; Kelley, Robert F.; Rodrigues, Maria Luísa; Snedecor, Brad; Covarrubias, M.A. Laredo; Velligan, Mark; Wong, Wai Lee; Rowland, Ann M.; Kotts, Claire E.; Carver, Monique E.; Yang, Maria; Bourell, James H.; Shepard, H M; Henner, Dennis J.

doi:10.1038/nbt0292-163

Cited by 270 publications

(118 citation statements)

References 38 publications

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“…First, the expression of MBP-scFvs may become the method-of choice for the ef®cient production of recombinant antibodies in bacteria. The production yields we report, 70-120 mg of puri®ed MBP-scFv from 2 g of wet bacterial paste exceed the best periplasmically expressed scFv 78 and parallels the production level we achieve from insoluble cytoplasmic inclusion bodies by matrix-assisted refolding. 79 It is conceivable that, with further optimization of growth and induction conditions (medium composition, inducer concentration, growth temperature), 7,42 our system can be made even more ef®cient.…”

Section: Discussionmentioning

confidence: 96%

Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies 1 1Edited by R. Huber

Bach

Mazor

Shaky

et al. 2001

Journal of Molecular Biology

166

119

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Recombinant single-chain antibodies (scFvs) that are expressed in the cytoplasm of cells are of considerable biotechnological and therapeutic potential. However, the reducing environment of the cytoplasm inhibits the formation of the intradomain disul®de bonds that are essential for correct folding and functionality of these antibody fragments. Thus, scFvs expressed in the cytoplasm are mostly insoluble and inactive.Here, we describe a general approach for stabilizing scFvs for ef®cient functional expression in the cell cytoplasm in a soluble, active form. The scFvs are expressed as C-terminal fusions with the Escherichia coli maltose-binding protein (MBP). We tested a large panel of scFvs that were derived from hybridomas and from murine and human scFv phage display and expression libraries by comparing their stability and functionality as un-fused versus MBP fused proteins. We found that MBP fused scFvs are expressed at high levels in the cytoplasm of E. coli as soluble and active proteins regardless of the redox state of the bacterial cytoplasm. In contrast, most un-fused scFvs can be produced (to much lower levels) in a functional form only when expressed in trxB À but not in trxB E. coli cells. We show that MBP-scFv fusions are more stable than the corresponding un-fused scFvs, and that they perform more ef®ciently in vivo as cytoplasmic intrabodies in E. coli. Thus, MBP seems to function as a molecular chaperone that promotes the solubility and stability of scFvs that are fused to it.

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Section: Discussionmentioning

confidence: 96%

Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies 1 1Edited by R. Huber

Bach

Mazor

Shaky

et al. 2001

Journal of Molecular Biology

166

119

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“…Although available data demonstrate that they greatly vary in tumor targeting efficiency (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40), direct comparisons are problematic because of the great differences among the various tumor models and antigens used.…”

mentioning

confidence: 99%

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

Kubetzko

Balic

Waibel

et al. 2006

Journal of Biological Chemistry

112

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A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185 HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

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“…For example, quantities of 1 to 2 g of soluble, assembled FabЈ fragments/liter have been demonstrated in the periplasm of Escherichia coli (3). 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).…”

mentioning

confidence: 99%

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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High Level Escherichia coli Expression and Production of a Bivalent Humanized Antibody Fragment

Cited by 270 publications

References 38 publications

Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies 1 1Edited by R. Huber

Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies 1 1Edited by R. Huber

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

Characterization of Humanized Antibodies Secreted byAspergillus niger

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