Isolation of antigen specific Llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae

Frenken, Leon; Linden, Richard H.J van der; Hermans, Pim; Bos, J. Wil; Ruuls, Robin; Geus, B. de; Verrips, C. Theo

doi:10.1016/s0168-1656(99)00228-x

Cited by 242 publications

(174 citation statements)

References 34 publications

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“…Upon DFO stimulation, stabilized HIF-1a translocates to the nucleus and induces transcription of target genes. 34 Indeed, we could visualize translocation of the mouse HIF-1a protein into the nucleus in DFOstimulated mouse NIH 3T3 fibroblasts (Figure 8c and d). Both the monovalent VHH AG1 and AG2 could detect a similar nuclear signal (data not shown).…”

Section: Endogenous Human Hif-1a Detection By Immunohistochemistrymentioning

confidence: 90%

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Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha

Groot

Verheesen

Westerlaken

et al. 2006

Laboratory Investigation

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Hypoxia triggers the transcription of genes responsible for cell survival via the key player transcription factor hypoxia-inducible factor 1alpha (HIF-1a). Overexpression of this protein has been implicated in cardiovascular disorders, carcinogenesis and cancer progression. For functional and diagnostic studies on the HIF-1a protein, we have identified single-domain antibody fragments directed against this protein by using a llama-derived nonimmune phage display library. This library displays the variable domains of the heavy-chain antibody subclass, found in these animals. Phage display selection with six recombinant HIF-1a proteins yielded five different antibody fragments. By epitope-mapping, we show that all five antibody fragments bind within the functionally important oxygen-dependent degradation domain of the HIF-1a protein. Two of these antibody fragments were engineered into bivalent antibodies that were able to detect human HIF-1a by immunohistochemistry, Western blotting and immunoprecipitation, and mouse HIF-1a by immunofluorescence and immunoprecipitation. These are the first single-domain antibody fragments that may be used in exploration of HIF-1a as a possible therapeutic target through molecular applications.

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Section: Endogenous Human Hif-1a Detection By Immunohistochemistrymentioning

confidence: 90%

“…This phage display library was generated essentially as described before. 34 Peripheral blood lymphocytes collected from the blood of eight nonimmunized llamas were used for mRNA extraction. cDNA was synthesized and VHH genes were PCR amplified introducing appropriate restriction sites for cloning.…”

Section: Selection Of Vhhmentioning

confidence: 99%

Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha

Groot

Verheesen

Westerlaken

et al. 2006

Laboratory Investigation

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“…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).…”

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

112

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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“…Антиген-связывающая область [46]. Одним из альтернативных источников получе-ния наноантител могут быть и трансгенные рас-тения [95].…”

Section: рисунок 1 схематичное изображение антител человека (а) хряunclassified

Single Domain Antibodies and Bioengineering Drugs on Their Basis: New Opportunities for Diagnostics and Therapy

Николаевна¹,

Vasilenko²,

Тиллиб³

et al. 2016

Med. immunol.

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Isolation of antigen specific Llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae

Cited by 242 publications

References 34 publications

Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha

Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha

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

Single Domain Antibodies and Bioengineering Drugs on Their Basis: New Opportunities for Diagnostics and Therapy

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