Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

Joosten, Vivi; Roelofs, Marc S.; Dries, Niels van den; Goosen, T.; Verrips, C. Theo; Hondel, Cees A. M. J. J. van den; Lokman, B. Christien

doi:10.1016/j.jbiotec.2005.06.034

Cited by 22 publications

(11 citation statements)

References 39 publications

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“…There is only one example of VHH production in filamentous fungi, which resulted in limited proteolytic degradation of the secreted product (Joosten et al 2005) because of the high levels of proteases secreted by filamentous fungi (Gerngross 2004). VHHs have also often been produced in the yeast Saccharomyces cerevisiae (Frenken et al 2000; Thomassen et al 2002; Van der Vaart 2002).…”

Section: Production In Microorganismsmentioning

confidence: 99%

Properties, production, and applications of camelid single-domain antibody fragments

Harmsen

Haard

2007

Appl Microbiol Biotechnol

683

537

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Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications.

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Section: Production In Microorganismsmentioning

confidence: 99%

Properties, production, and applications of camelid single-domain antibody fragments

Harmsen

Haard

2007

Appl Microbiol Biotechnol

683

537

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“…In Trichoderma reesei , 150 mg/L of a Fab fragment was obtained when both chains were fused with cellobiohydrolase I increasing yields 100-fold higher than with its natural signal peptide (121). A. awamori was used for the production of several scFvs, llama VHHs and antibody enzyme fusion proteins (117, 119, 122). A yield of 73.8 mg/L of an anti-EGFR-VHH was achieved in A. oryzae by using a Taka-amylase A signal sequence and 28 amino acids from the aminoterminal region of Rhizopus oryzae lipase (123).…”

Section: Eukaryotic Hosts Used For Antibody Productionmentioning

confidence: 99%

Expression of Recombinant Antibodies

Frenzel¹,

Hust²,

Schirrmann³

2013

Front. Immunol.

279

220

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Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

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“…To date, VHHs have been produced in E. coli , yeast, plants and mammalian cells [1], and there is only one example of VHH production in filamentous fungi [11]. It is important to highlight that for oral therapy a pre-requisite is the elimination of any living organism that could contaminate or replicate in the intestinal tract of patients, as well as the removal of any potential contaminating toxin that may aggravate the diarrhea.…”

Section: Introductionmentioning

confidence: 99%

Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo

et al. 2012

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BackgroundSingle-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs.ResultsHere we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea.ConclusionsOur results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule.

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Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

Cited by 22 publications

References 39 publications

Properties, production, and applications of camelid single-domain antibody fragments

Properties, production, and applications of camelid single-domain antibody fragments

Expression of Recombinant Antibodies

Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo

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