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Therapeutic Antibodies pp 187–216Cite as

Selection of Non-aggregating VH Binders from Synthetic VH Phage-Display Libraries

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 525))

Abstract

The particular interest in VH antibody fragments stems from the fact that they can rival their “naturally occurring” single-domain antibody (sdAb) counterparts (camelid VHHs and shark VNARs) with regard to such desirable characteristics as stability, solubility, expression, and ability to penetrate cryptic epitopes and outperform them in terms of less immunogenicity, a much valued property in human immunotherapy applications. However, human VHs are typically prone to aggregation. Various approaches for developing non-aggregating human VHs with binding specificities have relied on a combination of recombinant DNA technology and phage-display technology. VH gene libraries are constructed synthetically by randomizing the CDRs of a single VH scaffold fused to a gene encoding a phage coat protein. Recombinant phage expressing the resulting VH libraries in fusion with the pIII protein is propagated in Escherichia coli. Monoclonal phage displaying VHs with specificities for target antigens are isolated from the libraries by a process called panning. The exertion of stability pressure in addition to binding pressure during panning ensures that the isolated VH binders are also non-aggregating. The genes encoding the desired VHs selected from the libraries are packaged within the phage particles, linking genotype and phenotype, hence making possible the identification of the selected VHs through identifying its physically linked genotype. Here, we describe the application of recombinant DNA and phage-display technologies for the construction of a phage-displayed human VH library, the panning of the library against a protein, and the expression, purification, and characterization of non-aggregating VHs isolated by panning.

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Notes

  1. 1.

    This is National Research Council of Canada Publication 00000.

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Acknowledgments

The assistance of Rebecca To, Nathalie Gaudette, and Hong Tong-Sevinc with library construction, panning, protein expression, and purification is gratefully acknowledged. We thank Shakeeba Waseh for performing size-exclusion chromatography experiments. Requests for pMED1 vector should be addressed to Mehdi Arbabi-Ghahroudi.

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  1. National Research Council of Canada, Institute for Biological Sciences, Ottawa, Ontario, Canada

    Mehdi Arbabi-Ghahroudi, Roger MacKenzie & Jamshid Tanha

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  1. Mehdi Arbabi-Ghahroudi
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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Arbabi-Ghahroudi, M., MacKenzie, R., Tanha, J. (2009). Selection of Non-aggregating VH Binders from Synthetic VH Phage-Display Libraries. In: Dimitrov, A. (eds) Therapeutic Antibodies. Methods in Molecular Biology™, vol 525. Humana Press. https://doi.org/10.1007/978-1-59745-554-1_10

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