Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent

Immunohistochemistry of the PrP^Sc deposits in the cerebellum of a sCJD patient (upper three figures).

<p>Immunolabeling was performed with whole mAb V5B2, murine scFv (moScFv) and humanized scFv (huScFv) of V5B2. The arrow marks PrP^Sc plaques, while the triangle marks the diffuse, synaptic PrP^Sc deposition. On the lower three figures immunolabeling was performed on the cerebellum of the CJD negative patient.</p

Western blotting of the recombinant human PrP 23–226 (PrP226*) and of the recombinant human PrP23–231 (HuPrP).

<p>Reaction with the whole mAb V5B2 is compared to reactions with murine scFv (moScFv) and humanized scFv (huScFv) of V5B2. mAb 6H4 was used as a control antibody. Approximate molecular weights are in kilodaltons.</p

Amino acid sequence alignments.

<p>Alignment of light chain (Vl) and heavy chain (Vh) variable regions of the murine V5B2 scFv (moScFv), two humanized (LLHmut1, LLHmut2) and final humanized version (huScFv) of mAb V5B2 with human consensus sequences of light chain κ subgroup I (HhumκI) and heavy chain subgroup III (HumIII). The dashes represent unchanged amino acids. CDRs are underlined. Amino acids are numbered according to Kabat <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015783#pone.0015783-Kabat1" target="_blank">[36]</a>.</p

SDS-PAGE analyses of mouse (moScFv) and humanized (huScFv) scFv V5B2.

<p>Cell lysates were analyzed before (−) and after (+) IPTG induction, along with purified scFvs (e). Molecular masses of protein standards in kDa are indicated on the left. Similar electrophoretic pattern was obtained with all humanized scFv variants.</p

Identity and similarity scores of the V5B2 humanized variable domain frameworks with HhumκI and HumIII human antibody frameworks calculated by ClustalW.

<p><i>X</i> is the number of identical framework residues; <i>Y</i> is the total number of framework residues.</p

ELISA analysis of the antigen-binding efficiency of moScFv and huScFv.

<p>The absorbance at 405 nm is plotted against concentration of antibody fragment added to the wells coated with the P1 or P1Q peptide. The apparent affinity of moScFv in comparison with Fab V5B2 has been discussed previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015783#pone.0015783-krlj1" target="_blank">[31]</a>.</p