Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability

Abstract

Folding stabilities of camelized human antibody VH domains were studied through the determination of their melting points in thermodenaturation experiments. The melting point of a VH domain originating from a synthetic library of human VHs, which had been optimized for the use as small recognition units through the mimicking of camelid antibody heavy chains occurring naturally without light chain, was 56.6 degrees C compared with 71.2 degrees C of the original human VH. Its stability was improved (melting point 61.6 degrees C) through three mutations to mimic camelid VHs even further: Va137 was replaced by phenylalanine and two cysteines were introduced at position 33 and 100b. The resulting VH folded properly and formed a second intradomain disulphide between the extra cysteines. The new mutations were then built constitutively into a phage-display VH library, from which antigen-specific VHs were selected. Two were analysed for stability with melting points of 72.6 and 75.3 degrees C. Thus secondary camelization enabled the isolation of VHs with improved folding stabilities exceeding even that of the original human VH. This indicates an effect on folding stability for some mutations specific in the light chain lacking camelid heavy chains.

Generation of heavy-chain-only antibodies in mice

“…GS was generated because of later reports showing that MT mice produce some IgG, IgA, and IgE in the absence of membrane IgM (15)(16)(17), suggesting some B cells develop without IgM surface expression. Instead of mutating human VH domains to improve solubility (18,19), two llama VHHs were introduced. Camelid VHH contain characteristic amino acids at positions 42, 49, 50, and 52 (20,21).…”

“…Whilst it is preferable to avoid multiple copies on a single allele, it would be advantageous to have multiple alleles with a single copy of different VH regions to increase diversity. In such new loci, one can use either normally occurring (human) VH or VH engineered for increased solubility (18).…”

General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

“…Consequently, it is crucial to understand the influence of each of these framework-2 substitutions on the biophysical properties of Nanobodies to produce the most optimal single-domain antibody format. To date, most studies attempting to unravel the function of these VHH hallmark residues have focused on the partial camelization of conventional VH single domains to exploit the favorable properties of Nanobodies (12,37,47,48). This approach achieved only limited success as camelizing mutations on human or mouse VH was shown to be thermodynamically destabilizing due to framework deformations and did not completely eliminate the tendency to dimerize and aggregate (49 -51).…”

Design and testing of PCR primers for the construction of scFv libraries representing the immunoglobulin repertoire of rats

“…Immunoglobulin (Ig) proteins and genes have been extensively characterized from human and murine B cells (IMGT database, Montpellier, France (Giudicelli et al, 2004) and these models have become the primary source of monoclonal antibodies and recombinant antibodies for research and therapeutic applications. More recently, other immunoglobulin sources have been utilized, such as camel (Davies and Riechmann, 1996), rabbit (Ridder et al, 1995), sheep (Li et al, 2000), chicken (Foord et al, 2007), shark (Schluter et al, 2005), etc., since these sources have been found advantageous for particular applications. Technology exists to re-engineer these antibodies to be indistinguishable from human antibodies by grafting the CDR regions into a human antibody framework (Jones et al, 1986), thus making it easier to develop antibodies using different animal model for human purposes.…”