Selection of non-aggregating VH binders from synthetic VH phase-display libraries

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TypeBook Chapter
Book titleTherapeutic Antibodies: Methods and Protocols
Series titleMethods in Molecular Biology; Volume 525
SubjectAnimals; Antibodies; antibody; Antibody Affinity; ANTIGEN; Antigens; Bacteriophages; binding; Canada; CHAIN; CHAINS; chemistry; Chromatography,Gel; Cloning,Molecular; DNA; Enzyme-Linked Immunosorbent Assay; epitope; Epitopes; Escherichia; Escherichia coli; ESCHERICHIA-COLI; EXPRESSION; GENE; Gene Library; Genes; Hot Temperature; Human; Humans; IDENTIFICATION; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; immunology; isolation & purification; Libraries; Methods; Molecular Biology; peptide; Peptide Library; Phenotype; protein; Protein Denaturation; Protein Structure,Quaternary; REGION; SELECTION; Solubility; SPECIFICITY; stability; Sus scrofa; synthetic; TARGET
AbstractThe 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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PublisherHuman Press
AffiliationNational Research Council Canada (NRC-CNRC); NRC Institute for Biological Sciences
Peer reviewedYes
NPARC number15329262
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Record identifier8e6b00e2-ccee-494a-a8e8-75bd271bf2c6
Record created2010-05-21
Record modified2016-05-09
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