Recent advances in the large-scale production of antibody fragments using lower eukaryotic microorganisms

Frenken, Leon; Hessing, J. G. M.; Hondel, Cees A. M. J. J. van den; Verrips, C. Theo

doi:10.1016/s0923-2494(98)80011-4

Cited by 54 publications

(24 citation statements)

References 58 publications

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“…Given the unique properties attributed to VHH, in terms of solubility, thermal and chemical stability, and high expression levels leading to a low production cost (20,26,64,81), neutralizing VHH might prove useful in a number of applications, for example, as candidate HIV-1 microbicides as well as antiretroviral drugs or prophylactics. Topical application of MAb b12 has been shown to protect macaques from infection after vaginal challenge with SHIV, which supports the potential use of antibodies for topical prevention of HIV-1 transmission (82).…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, they have been shown to have a preference for cleft recognition and for binding into active sites (16,41). The VHH domain can be easily cloned and expressed to high levels in bacteria and yeast (26,27). This notion, together with advantageous characteristics in terms of stability and solubility (20,64,81), has led to successful development of camelid VHH in a number of applications against a range of biological targets (2,13,14,19,21,57,58,69,83,84), including neutralization of rotavirus (28,60).…”

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confidence: 99%

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Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

Forsman

Beirnaert

Aasa-Chapman

et al. 2008

J Virol

111

138

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Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B /C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC 50 ) and IC 90 values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

Forsman

Beirnaert

Aasa-Chapman

et al. 2008

J Virol

111

138

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“…A cleavage site (Lys-Arg) recognized by the Golgi apparatus serine proteinase KexB (13) is often used to allow release of the mammalian protein during the secretion process (reviewed in reference 10). 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).…”

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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“…A cleavage site (Lys Arg "KR") recognized by the Golgi serine proteinase Kex2 (Jalving et al, 2000) is often used to allow release of the authentic mammalian protein during the secretion process (reviewed in Gouka et al, 1997) (Figure 2). This approach has been used by Frenken et al (1998) for the production of an scFv in A. niger. Work in T. reesei has demonstrated production and assembly of a Fab fragment.…”

Section: Statement Of the Problem Studiedmentioning

confidence: 99%

Develop Systems for Manufacturing 100,000,000 Doses of an Emergency Pharmaceutical (e.g. Vaccine or Monoclonal Antibody) Within 2 Months of Product Identification

Baldwin¹,

Estell²,

Fung³

et al. 2006

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Recent advances in the large-scale production of antibody fragments using lower eukaryotic microorganisms

Cited by 54 publications

References 58 publications

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

Characterization of Humanized Antibodies Secreted byAspergillus niger

Develop Systems for Manufacturing 100,000,000 Doses of an Emergency Pharmaceutical (e.g. Vaccine or Monoclonal Antibody) Within 2 Months of Product Identification

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